Protokoll der IV. Sitzung

Ohne Zusammenfassung     

From evil to good: a cytolysin in vaccine development

Current vaccination strategies mainly target antigens into the phagosomal, major histocompatibility complex class II antigen-processing pathway and thus lead predominantly to humoral immune responses. The elicitation of cytotoxic T-cell responses instead requires introduction of antigens into the cytosol of professional antigen-presenting cells (APCs). The intracellular bacterium Listeria monocytogenes gains access to the host cell cytosol by means of a cytolysin, listeriolysin O. Vaccine researchers have successfully employed listeriolysin in novel vaccination approaches to provide access to the cytosol of professional APCs for purified protein antigens, attenuated bacterial vaccine strains, DNA vaccines and liposome contents.     

Identification of tissue-specific microRNAs from mouse

MicroRNAs (miRNAs) are a new class of noncoding RNAs, which are encoded as short inverted repeats in the genomes of invertebrates and vertebrates. It is believed that miRNAs are modulators of target mRNA translation and stability, although most target mRNAs remain to be identified. Here we describe the identification of 34 novel miRNAs by tissue-specific cloning of approximately 21-nucleotide RNAs from mouse. Almost all identified miRNAs are conserved in the human genome and are also frequently found in nonmammalian vertebrate genomes, such as pufferfish. In heart, liver, or brain, it is found that a single, tissue-specifically expressed miRNA dominates the population of expressed miRNAs and suggests a role for these miRNAs in tissue specification or cell lineage decisions. Finally, a miRNA was identified that appears to be the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA.     

Combined Ki-67 and feulgen stain for morphometric determination of the Ki-67 labelling index

In this note we present a combined Ki-67 and Feulgen stain for morphometric determination of the Ki-67 labelling index. The immunohistochemical part of this double staining technique is based on the alkaline-phosphatase-anti-alkaline-phosphatase (APAAP) method, visualizing the enzyme activity by the nitro-blue-tetrazolium chloride (NBT)/bromo-chloro-3-indolyl-phosphate (BCIP) technique. The NBT/BCIP complex resists the hydrolytic activity of the Feulgen stain. The staining method presented allows semi-automatic determination of both the total nucleus-area as well as the Ki-67 positive nucleus-area using a morphometric computer system. The Ki-67 labelling index thus achieved is based on the relative nuclear area of Ki-67 positive nuclei and is clearly more precise and efficient than the counting method using an ocular grid.     

Bacteriorhodopsin,boundary lipid and protein conformers. A spin label study

A spin label study, as a function of temperature, has been made with the bacteriorhodopsin membrane using a stearic acid spin label. The ESR spectra show a strong variation with temperature and the presence of isosbestic points. The spectra are interpreted as indicating the presence of a two-component system with an activation energy (approx. 14 kcal/mol) corresponding to a protein conformational change. This activation energy is similar to that deduced from recent flash photolysis studies.It is concluded that the spin label is sensitive to the temperature-dependent protein conformational change in this membrane system.     

Chemical crosslinking of different forms of the simian virus 40 large T antigen using bifunctional reagents

Chemical crosslinking was used for a direct analysis of the different forms of large tumor (T) antigen, the simian virus 40 A gene product. The first subclass, sedimenting at 14-16S, is composed of monomeric to tetrameric units, whereas the second, sedimenting at 5-6S, only contains dimers and monomers of T. The occurrence of oligomeric structures of T in solution which are higher than dimers suggests the possibility of direct binding of such trimers or tetramers to the origin of replication of the viral DNA as an alternative to the formation of these structures by aggregation of bound dimers or monomers after their sliding along the DNA.     

Gene delivery to respiratory epithelial cells by magnetofection

BACKGROUND: For the topical application of DNA vector complexes to the airways, specific extracellular barriers play a major role. In particular, short contact time of complexes with the cell surface caused by the mucociliary clearance hinders cellular uptake of complexes. The aim of this study was to evaluate the ability of magnetofection, a technique based on the principle of magnetic drug targeting, to overcome these barriers in comparison with conventional nonviral gene transfer methods such as lipofection and polyfection. METHODS: Experiments were carried out on permanent (16HBE14o-) and primary airway epithelial cells (porcine and human), and native porcine airway epithelium ex vivo. Transfection efficiency and dose-response relationship of magnetofection were examined by luciferase reporter gene expression. Sedimentation patterns and uptake of gene transfer complexes were characterized by fluorescence and electron microscopy, respectively. RESULTS: We show that (i) application of a magnetic field allows the magnetofectins to sediment and to enrich at the cell surface within a few minutes, (ii) magnetofection bears an improved dose-response relationship, (iii) magnetofection enhances transfection efficiency in both, permanent and primary airway epithelial cells, and (iv) magnetofection leads to significant transgene expression at very short incubation times in an ex vivo airway epithelium organ model. CONCLUSIONS: Magnetofection provides a potential novel method, which may overcome fundamental limitations of nonviral gene transfer to the airways. Due to the accelerated enrichment at the cell surface it may be of major interest for in vivo applications, where long-term incubation times at the target tissue are hardly achievable.     

Response of bacterial community during bioremediation of an oil-polluted soil

AIM: To study the response of the bacterial community to bioremediation of a soil with an aged contamination of crude oil. METHODS AND RESULTS: The bacterial community in laboratory soil columns during a 72-day biostimulation treatment was followed by analysing the number of total cultivable hydrocarbon-degrading bacteria, soil respiratory activity and the 16S-23S rDNA internal transcribed spacer homoduplex heteroduplex polymorphisms (ITS-HHP) of total soil bacterial DNA. ITS-HHP permits an estimate of both length and sequence polymorphism in a 16S-23S rDNA spacer population, using to advantage the homoduplex and heteroduplex fragments that are generated during PCR. The treatment, made by air sparging and biostimulation with a mineral nutrient and surfactant solution, resulted in a 39.5% decrease of the total hydrocarbon content. Within 4 days of treatment onset the bacterial community underwent a first phase of activation that led to a substantial increase in the observable diversity. Subsequently, after a 12-day period of stability, another activation phase was observed with further shifts of the community structure and an increase in the abundance and diversity of catechol-2,3-dioxygenase (C23O) genes. CONCLUSIONS: The overall data suggest an important contribution of uncultivable bacteria to the soil bioremediation, since, during the second activation phase, the increases of the respiratory activity, bacterial diversity and C23O gene abundance and diversity were not accompanied by a corresponding increase of the cultivable bacteria number. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that successive phases of activation of bacterial populations occur during a bioremediation treatment of oil-polluted soil.