Antibodies Raised Against Synthetic Peptides React with Choline Acetyltransferase in Various Immunoassays and in Immunohistochemistry

Abstract: Antisera were raised in rabbits against five synthetic peptides. These peptides have been identified as potentially antigenic epitopes from the sequence of porcine choline acetyltransferase (ChAT) using primary and secondary structure analysis. All five antisera recognized immunoaffinity-purified antigen from porcine brain in an ELISA and on western blots. Four antisera recognized ChAT on dot blots, and another four antisera reacted with native and degraded enzyme in a sandwich ELISA using monoclonal antibodies as the capture antibody. One peptide antiserum was of similar avidity in this sandwich ELISA as a polyclonal antibody raised against immunoaffinity-purified ChAT. The same antiserum reacted with the enzyme from human placenta in an ELISA and on western and dot blots and recognized ChAT in rat, primate, and human neurons. Thus, a single peptide (amino acids 168- 189) provides the means for easy, reliable, and reproducible generation of antibodies against ChAT suitable for replacing conventional polyclonal and monoclonal antibodies.     

Assessing Conservation Action for Substitution of Missing Dynamics on Former Military Training Areas in Central Europe

Disproportionately large numbers of threatened and endangered species and unusually high biodiversity have been documented on former military training areas. However, throughout the world, nature protection areas on former military grounds are faced with the dilemma of preserving ecosystems that must change. In central Europe, many of these areas are characterized by dry acidic grasslands on sandy soils. However, these resource-limited ecosystems are faced with two interacting threats: missing disturbance dynamics and increasing atmospheric nitrogen deposition from urban agglomerations. The goal of this study was to assess the effectiveness of conservation action for substitution of missing dynamics. Impacts on plant species assembly, performance of functional groups, soil nutrient availability, and successional trajectory are analyzed. Of particular concern is mimicking beneficial effects of military maneuver on pioneer species such as Corynephorus canescens , which is endangered in many countries. In a controlled field experiment, we manipulated mechanical disturbances representing conservation measures—harrowing, topsoil removal, tilling, and tank driving. Our findings suggest that only topsoil removal and tank driving effectively remove organic material and create self-sustaining vegetation dynamics. Topsoil removal affects both filters in community assembly: the abiotic filter related to soil resources and the biotic filter related to competition between species. We further demonstrate that plant functional groups based on growth form show different rates of recruitment after disturbance with graminoids-dominating initial dynamics. This study provides basic information on effective conservation strategies for dry acidic grasslands on former military training areas.     

Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10⁻⁶ to 5.8 × 10⁻⁷ transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10⁻⁹). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10⁻¹⁰), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10⁻¹¹. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

Habitat suitability models for the conservation of thermophilic grasshoppers and bush crickets—simple or complex?

One goal of conservation biology is the assessment of effects of land use change on species distribution. One approach for identifying the factors, which determine habitat suitability for a species are statistical habitat distribution models. These models are quantitative and can be used for predictions in management scenarios. However, they often have one major shortcoming, which is their complexity. This means that they need several, often costly-to-determine parameters for predictions of species occurrence. We first used habitat suitability models to investigate and determine habitat preferences of three different Orthoptera species. Second, we compared the predictive powers of simple habitat suitability models considering only the ‘habitat type’ as predictor with more complex models taking different habitat factors into account. We found that the habitat type is the most reliable and robust factor, which determines the occurrence of the species studied. Thus, analyses of habitat suitability can easily be carried out on the basis of existing vegetation maps for the conservation of the three species under study. Our results can serve as a basis for the estimation of spatio-temporal distribution and survival probabilities of the species studied and might also be valuable for other species living in dry grasslands.     

Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria

During cultivation under storage conditions with BG11 medium containing acetate as a carbon source, Synechocystis sp. PCC6803 accumulated poly(3-hydroxybutyrate) up to 10% (w/w) of the cell dry weight. Our analysis of the complete Synechocystis sp. PCC6803 genome sequence, which had recently become available, revealed that not only the open reading frame slr1830 (which was designated as phaC) but also the open reading frame slr1829, which is located colinear and upstream of phaC, most probably represent a polyhydroxyalkanoic acid (PHA) synthase gene. The open reading frame slr1829 was therefore designated as phaE. The phaE and phaC gene products exhibited striking sequence similarities to the corresponding PHA synthase subunits PhaE and PhaC of Thiocystis violacea, Chromatium vinosum, and Thiocapsa pfennigii. The Synechocystis sp. PCC6803 genes were cloned using PCR and were heterologously expressed in Escherichia coli and in Alcaligenes eutrophus. Only coexpression of phaE and phaC partially restored the ability to accumulate poly(3-hydroxybutyrate) in the PHA-negative mutant A. eutrophus PHB^–4. These results confirmed our hypothesis that coexpression of the two genes is necessary for the synthesis of a functionally active Synechocystis sp. PCC6803 PHA synthase. PHA granules were detected by electron microscopy in these cells, and the PHA-granule-associated proteins were studied. Western blot analysis of Synechocystis sp. PCC6803 crude cellular extracts and of granule-associated proteins employing antibodies raised against the PHA synthases of A. eutrophus (PhaC) and of C. vinosum (PhaE and PhaC) revealed no immunoreaction. Received: 11 March 1998 / Accepted: 2 June 1998     

Structural Requirements for the Procoagulant Activity of Nucleic Acids

Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.     

Absence of Foxp3+ Regulatory T Cells during Allergen Provocation Does Not Exacerbate Murine Allergic Airway Inflammation

Regulatory T cells (Tregs) play a non-redundant role in maintenance of immune homeostasis. This is achieved by suppressing both, priming of naïve cells and effector cell functions. Although Tregs have been implicated in modulating allergic immune responses, their influence on distinct phases of development of allergies remains unclear. In this study, by using bacterial artificial chromosome (BAC)-transgenic Foxp3-DTR (DEREG) mice we demonstrate that the absence of Foxp3⁺ Tregs during the allergen challenge surprisingly does not exacerbate allergic airway inflammation in BALB/c mice. As genetic disposition due to strain specificity may contribute significantly to development of allergies, we performed similar experiment in C57BL/6 mice, which are less susceptible to allergy in the model of sensitization used in this study. We report that the genetic background does not influence the consequence of this depletion regimen. These results signify the temporal regulation exerted by Foxp3⁺ Tregs in limiting allergic airway inflammation and may influence their application as potential therapeutics.     

Lysozyme-lysozyme self-interactions as assessed by the osmotic second virial coefficient: Impact for physical protein stabilization

The purpose of the presented study is to understand the physicochemical properties of proteins in aqueous solutions in order to identify solution conditions with reduced attractive protein-protein interactions, to avoid the formation of protein aggregates and to increase protein solubility. This is assessed by measuring the osmotic second virial coefficient (B²²), a parameter of solution non-ideality, which is obtained using self-interaction chromatography. The model protein is lysozyme. The influence of various solution conditions on B²² was investigated: protonation degree, ionic strength, pharmaceutical relevant excipients and combinations thereof. Under acidic solution conditions B²² is positive, favoring protein repulsion. A similar trend is observed for the variation of the NaCl concentration, showing that with increasing the ionic strength protein attraction is more likely. B²² decreases and becomes negative. Thus, solution conditions are obtained favoring attractive protein-protein interactions. The B²² parameter also reflects, in general, the influence of the salts of the Hofmeister series with regard to their salting-in/salting-out effect. It is also shown that B²² correlates with protein solubility as well as physical protein stability.     

Going live: A comparative analysis of the suitability of the RFP derivatives RedStar,mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites

Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-generation red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.