Real-time PCR method for detection of pathogenic Yersinia enterocolitica in food

The current methods for the detection of pathogenic Yersinia enterocolitica bacteria in food are time consuming and inefficient. Therefore, we have developed and evaluated in-house a TaqMan probe-based real-time PCR method for the detection of this pathogen. The complete method comprises overnight enrichment, DNA extraction, and real-time PCR amplification. Also included in the method is an internal amplification control. The selected primer-probe set was designed to use a 163-bp amplicon from the chromosomally located gene ail (attachment and invasion locus). The selectivity of the PCR method was tested with a diverse range (n = 152) of related and unrelated strains, and no false-negative or false-positive PCR results were obtained. The sensitivity of the PCR amplification was 85 fg purified genomic DNA, equivalent to 10 cells per PCR tube. Following the enrichment of 10 g of various food samples (milk, minced beef, cold-smoked sausage, fish, and carrots), the sensitivity ranged from 0.5 to 55 CFU Y. enterocolitica. Good precision, robustness, and efficiency of the PCR amplification were also established. In addition, the method was tested on naturally contaminated food; in all, 18 out of 125 samples were positive for the ail gene. Since no conventional culture method could be used as a reference method, the PCR products amplified from these samples were positively verified by using conventional PCR and sequencing of the amplicons. A rapid and specific real-time PCR method for the detection of pathogenic Y. enterocolitica bacteria in food, as presented here, provides a superior alternative to the currently available detection methods and makes it possible to identify the foods at risk for Y. enterocolitica contamination.     

Biosynthesis of the Metalloclusters of Molybdenum Nitrogenase

Summary: Nitrogenase catalyzes a key step in the global nitrogen cycle, the nucleotide-dependent reduction of atmospheric dinitrogen to bioavailable ammonia. There is a substantial amount of interest in elucidating the biosynthetic mechanisms of the FeMoco and the P-cluster of nitrogenase, because these clusters are not only biologically important but also chemically unprecedented. In this review, we summarize the recent advances in this research area, with an emphasis on our work that aims at providing structural and spectroscopic insights into the assembly of these complex metalloclusters.     

No evidence for DUP25 in patients with panic disorder using a quantitative real-time PCR approach

A duplication of chromosome 15q24-q26 (DUP25) has been reported to be associated with anxiety disorders. We tested for the presence of DUP25 in a sample of 50 patients with panic disorder and 50 controls using a quantitative real-time PCR approach. Contrary to the original finding, our results were compatible with the absence of DUP25, and no significant difference could be detected between patients and controls (P=1.0). Thus, our study does not support the hypothesis of an involvement of DUP25 in panic disorder.     

Tuberous sclerosis genes regulate cellular 14-3-3 protein levels

The genes TSC1, encoding hamartin, and TSC2, encoding tuberin are responsible for tuberous sclerosis. This autosomal dominant tumor suppressor gene syndrome affects about 1 in 6000 individuals. A variety of tumors characteristically occur in different organs of tuberous sclerosis patients and are believed to result from defects in cell cycle/cell size control. We performed a proteomics approach of two-dimensional gel electrophoresis with subsequent mass spectrometrical identification of protein spots after ectopic overexpression of human TSC1 or TSC2. We found the cellular levels of four isoforms of the 14-3-3 protein family, 14-3-3 gamma, 14-3-3, 14-3-3 sigma, and 14-3-3 zeta, to be regulated by the two tuberous sclerosis gene products. In the same experiments the protein levels of keratin 7, capZ alpha-1 subunit, ezrin, and nedasin were not affected by ectopic TSC1 or TSC2. Western blot analyses confirmed the deregulation of 14-3-3 proteins upon ectopic overexpression of TSC1 and TSC2. A TSC1 mutant not encoding the transmembrane domain and the tuberin-binding domain but harbouring most of the coiled-coil region and the ERM protein interaction domain of hamartin did not affect 14-3-3 protein levels. The here presented findings suggest that deregulation of 14-3-3 protein amounts might contribute to the development of tumors in tuberous sclerosis patients. These data provide important new insights into the molecular development of this disease especially since both, the TSC genes and the 14-3-3 proteins, are known to be involved in mammalian cell cycle control.     

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.     

Regulation of differential pro- and anti-apoptotic signaling by glucocorticoids

More than a quarter of a century ago, the phenomenon of glucocorticoid-induced apoptosis in the majority of hematological cells was first recognized. More recently, glucocorticoid-induced antiapoptotic signaling associated with apoptosis resistance has been identified in cells of epithelial origin, most of malignant solid tumors and some other tissues. Despite these huge amount of data demonstrating differential pro- and anti-apoptotic effects of glucocortioids, the underlying mechanisms of cell type specific glucocorticoid signaling are just beginning to be described. This review summarizes our present understanding of cell type-specific pro- and anti-apoptotic signaling induced by glucocorticoids. In the first section we give a summary and update of known glucocorticoid-induced pathways mediating apoptosis in hematological cells. We shortly introduce mechanisms of glucocorticoid resistance of hematological cells. We highlight and discuss the emerging molecular evidence of a general induction of survival signaling in epithelial cells and carcinoma cells by glucocorticoids. We provide a model for glucocorticoid-induced resistance in cells growing in a tissue formation. Thus, attachment to the extracellular matrix and cell-cell contacts typical for e.g. epithelial and tumor cells may be crucially involved in switching the balance of several interacting pathways to survival upon treatment with glucocorticoids.