Elevated serum levels of heat shock protein 70 can be detected after radiofrequency ablation

Due to their adjuvant effect and their ability to chaperone tumor-associated peptides, heat shock proteins constitute a potent alarm signal for the immune system and can lead to activation of anti-tumor T-cell immunity. Radiofrequency ablation has been reported to induce heat shock protein expression especially that of heat shock protein 70 in sublethally damaged tumor cells. In this study, we evaluated the release of heat shock protein 70 into the serum of cancer-bearing patients directly after radiofrequency ablation. Sera of 22 patients undergoing radiofrequency ablation for the treatment of primary and secondary malignancies of the liver, kidney, and lung, as well as control sera of 20 patients undergoing diagnostic liver biopsy were analyzed using a manufactured heat shock protein 70 ELISA. A significant increase in serum levels of heat shock protein 70 was detectable in the patient cohort 1 day after radiofrequency ablation. More than a twofold increase was observed in nine out of 22 patients, which tended to correlate with favorable clinical outcome. No patient of the control group revealed a comparable increase. Radiofrequency ablation can lead to a release of heat shock protein 70 into the serum, which is transiently detectable 1 day after treatment. Elevated heat shock protein 70 serum levels may constitute a biomarker for favorable clinical outcome.     

Influence of growth temperature on the production of antibody Fab fragments in different microbes: a host comparative analysis

Microorganisms encounter diverse stress conditions in their native habitats but also during fermentation processes, which have an impact on industrial process performance. These environmental stresses and the physiological reactions they trigger, including changes in the protein folding/secretion machinery, are highly interrelated. Thus, the investigation of environmental factors, which influence protein expression and secretion is still of great importance. Among all the possible stresses, temperature appears particularly important for bioreactor cultivation of recombinant hosts, as reductions of growth temperature have been reported to increase recombinant protein production in various host organisms. Therefore, the impact of temperature on the secretion of proteins with therapeutic interest, exemplified by a model antibody Fab fragment, was analyzed in five different microbial protein production hosts growing under steady-state conditions in carbon-limited chemostat cultivations. Secretory expression of the heterodimeric antibody Fab fragment was successful in all five microbial host systems, namely Saccharomyces cerevisiae, Pichia pastoris, Trichoderma reesei, Escherichia coli and Pseudoalteromonas haloplanktis. In this comparative analysis we show that a reduction of cultivation temperature during growth at constant growth rate had a positive effect on Fab 3H6 production in three of four analyzed microorganisms, indicating common physiological responses, which favor recombinant protein production in prokaryotic as well as eukaryotic microbes.     

Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.     

Recombinant protein production in yeasts

Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.     

Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences

Organisms living in or on the sediment layer of water bodies constitute the benthos fauna, which is known to harbour a large number of species of diverse taxonomic groups. The benthos plays a significant role in the nutrient cycle and it is, therefore, of high ecological relevance. Here, we have explored a DNA-taxonomic approach to access the meiobenthic organismic diversity, by focusing on obtaining signature sequences from a part of the large ribosomal subunit rRNA (28S), the D3-D5 region. To obtain a broad representation of taxa, benthos samples were taken from 12 lakes in Germany, representing different ecological conditions. In a first approach, we have extracted whole DNA from these samples, amplified the respective fragment by PCR, cloned the fragments and sequenced individual clones. However, we found a relatively large number of recombinant clones that must be considered PCR artefacts. In a second approach we have, therefore, directly sequenced PCR fragments that were obtained from DNA extracts of randomly picked individual organisms. In total, we have obtained 264 new unique sequences, which can be readily placed into taxon groups, based on phylogenetic comparison with currently available database sequences. The group with the highest taxon abundance were nematodes and protozoa, followed by chironomids. However, we find also that we have by far not exhausted the diversity of organisms in the samples. Still, our data provide a framework within which a meiobenthos DNA signature sequence database can be constructed, that will allow to develop the necessary techniques for studying taxon diversity in the context of ecological analysis. Since many taxa in our analysis are initially only identified via their signature sequences, but not yet their morphology, we propose to call this approach 'reverse taxonomy'.     

An invasive lineage of sculpins, Cottus sp. (Pisces, Teleostei) in the Rhine with new habitat adaptations has originated from hybridization between old phylogeographic groups

Fish abundance surveys in the Rhine system have shown in the past two decades that there is a rapid upriver invasion of a freshwater sculpin of the genus Cottus. These fish are found in habitats that are atypical for the known species Cottus gobio, which is confined to small cold streams within the Rhine drainage. Phylogeographic analysis based on mitochondrial haplotypes and diagnostic single nucleotide polymorphisms indicates that the invasive sculpins are hybrids between two old lineages from the River Scheldt drainage and the River Rhine drainage, although it is morphologically more similar to the Scheldt sculpins. Most importantly, however, the invasive population possesses a unique ecological potential that does not occur in either of the source populations from the Rhine or the Scheldt, which allows the colonization of new habitats that have previously been free of sculpins. Microsatellite analysis shows that the new lineage is genetically intermediate between the old lineages and that it forms a distinct genetic group across its whole expansion range. We conclude that hybridization between long separated groups has lead to the fast emergence of a new, adaptationally distinct sculpin lineage.     

A segmentation gene in tribolium produces a polycistronic mRNA that codes for multiple conserved peptides

Segmentation genes in insects are required for generating the subdivisions of the early embryo. We describe here a new member of the gap family of segmentation genes in the flour beetle Tribolium, mille-pattes (mlpt). mlpt knockdown leads to transformation of the abdominal segments into thoracic segments, providing embryos with up to ten pairs of legs. We show that there are crossregulatory interactions between mlpt and the known gap genes in Tribolium, suggesting that mlpt is itself a gap gene. The mlpt gene reveals an unusual structure, as it encodes a polycistronic mRNA that codes for four peptides. mlpt appears to be the prototype of this previously unknown gene structure in eukaryotes, as we find homologous genes with the same polycistronic arrangement in other insect genomes as well.