Highly Sensitive Matrix-Independent Quantification of Major Food Allergens Peanut and Soy by Competitive Real-Time PCR Targeting Mitochondrial DNA.

The development of two competitive real-time PCR assays for the quantitative detection of trace amounts of two major food allergens, peanut and soybean, is reported. In order to achieve very low detection levels for both allergens, we established PCR primers and probes targeting mitochondrial DNA sequences. We were able to demonstrate that this approach led to an increase in detection sensitivity in the range of at least 1 order of magnitude compared with published assays targeting nuclear DNA.

Calcium-release channels in paramecium. Genomic expansion, differential positioning and partial transcriptional elimination.

The release of Ca²⁺ from internal stores is a major source of signal Ca²⁺ in almost all cell types. The internal Ca²⁺ pools are activated via two main families of intracellular Ca²⁺-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP₃) receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence.

Novel types of Ca2+ release channels participate in the secretory cycle of Paramecium cells.

A database search of the Paramecium genome reveals 34 genes related to Ca(2+)-release channels of the inositol-1,4,5-trisphosphate (IP(3)) or ryanodine receptor type (IP(3)R, RyR). Phylogenetic analyses show that these Ca(2+) release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP(3)Rs and RyRs. We characterize here the P.

Pharmacology of ciliated protozoa--drug (in)sensitivity and experimental drug (ab)use.

Most data on the effects of drugs as inhibitors, modulators, or stimulators have been collected with higher eukaryotic, mainly mammalian cells. Although in cell biological experiments with lower eukaryotes, including ciliates, the same drugs have frequently been applied, many results remained questionable for several reasons. Most drugs had to be used in unusually high concentrations.

An Ins(1,4,5)P3 receptor in Paramecium is associated with the osmoregulatory system.

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat.

Human Mcm proteins at a replication origin during the G1 to S phase transition.

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication.

The origin recognition complex marks a replication origin in the human TOP1 gene promoter.

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo.

Identification of a binding region for human origin recognition complex proteins 1 and 2 that coincides with an origin of DNA replication.

We investigated the binding regions of components of the origin recognition complex (ORC) in the human genome. For this purpose, we performed chromatin immunoprecipitation assays with antibodies against human Orc1 and Orc2 proteins. We identified a binding region for human Orc proteins 1 and 2 in a <1-kbp segment between two divergently transcribed human genes.