The BRCA1 Variant p.Ser36Tyr Abrogates BRCA1 Protein Function and Potentially Confers a Moderate Risk of Breast Cancer

The identification of variants of unknown clinical significance (VUS) in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.     

Synthesis and In vivo anti-inflammatory activity of long-chain 2-amino-alcohols

The synthesis of optically pure long-chain 2-amino-alcohols and 1-O-dodecyl-2-deoxy-2-amino-sn-glycerol was carried out starting from L- or D-Boc-Ser(OBn)-ol by oxidation and consecutive Wittig reaction or etherification reaction. 2-Amino-oleyl alcohol was synthesized by reduction of the corresponding 2-amino-oleic acid. All the long chain amino-alcohols presented interesting inhibition of carrageenin-induced paw edema in rats (ED(50) from 0.017 to 0.010 mmol/kg).     

R proteins as fundamentals of plant innate immunity

Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader’s molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity.     

Reactive oxygen species production by forward and reverse electron fluxes in the mitochondrial respiratory chain

Reactive oxygen species (ROS) produced in the mitochondrial respiratory chain (RC) are primary signals that modulate cellular adaptation to environment, and are also destructive factors that damage cells under the conditions of hypoxia/reoxygenation relevant for various systemic diseases or transplantation. The important role of ROS in cell survival requires detailed investigation of mechanism and determinants of ROS production. To perform such an investigation we extended our rule-based model of complex III in order to account for electron transport in the whole RC coupled to proton translocation, transmembrane electrochemical potential generation, TCA cycle reactions, and substrate transport to mitochondria. It fits respiratory electron fluxes measured in rat brain mitochondria fueled by succinate or pyruvate and malate, and the dynamics of NAD(+) reduction by reverse electron transport from succinate through complex I. The fitting of measured characteristics gave an insight into the mechanism of underlying processes governing the formation of free radicals that can transfer an unpaired electron to oxygen-producing superoxide and thus can initiate the generation of ROS. Our analysis revealed an association of ROS production with levels of specific radicals of individual electron transporters and their combinations in species of complexes I and III. It was found that the phenomenon of bistability, revealed previously as a property of complex III, remains valid for the whole RC. The conditions for switching to a state with a high content of free radicals in complex III were predicted based on theoretical analysis and were confirmed experimentally. These findings provide a new insight into the mechanisms of ROS production in RC.     

Microbial eukaryote life in the new hypersaline deep-sea basin Thetis

Only recently, a novel anoxic hypersaline (thalassic) basin in the eastern Mediterranean was discovered at a depth of 3,258 m. The halite-saturated brine of this polyextreme basin revealed one of the highest salt concentrations ever reported for such an environment (salinity of 348‰). Using a eukaryote-specific probe and fluorescence in situ hybridization, we counted 0.6 × 10⁴ protists per liter of anoxic brine. SSU rRNA sequence analyses, based on amplification of environmental cDNA identified fungi as the most diverse taxonomic group of eukaryotes in the brine, making deep-sea brines sources of unknown fungal diversity and hotspots for the discovery of novel metabolic pathways and for secondary metabolites. The second most diverse phylotypes are ciliates and stramenopiles (each 20%). The occurrence of closely related ciliate sequences exclusively in other Mediterranean brine basins suggests specific adaptations of the respective organisms to such habitats. Betadiversity-analyses confirm that microeukaryote communities in the brine and the interface are notably different. Several distinct morphotypes in brine samples suggest that the rRNA sequences detected in Thetis brine can be linked to indigenous polyextremophile protists. This contradicts previous assumptions that such extremely high salt concentrations are anathema to eukaryotic life. The upper salinity limits for eukaryotic life remain unidentified.     

Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman

Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR) and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ～3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV) while the activity of the α1 isoform decreased (-4.4 mV). Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM), measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63) and Ser(68). Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.     

A master switch couples Mg²⁺-assisted catalysis to domain motion in B. stearothermophilus tryptophanyl-tRNA Synthetase

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Highlights? Combinatorial mutagenesis at four remote sites shifts reaction coordinate stabilities ? Cooperative action of all four sites accounts for the entire catalytic role of Mg2⁺ ? Catalysis of ATP utilization by Mg2⁺ occurs if, and only if, the conformation changes ? This type of allosteric effect may explain vectorial coupling in transducing NTPases     

Activity of neuromodulatory neurones during stepping of a single insect leg

Octopamine plays a major role in insect motor control and is released from dorsal unpaired median (DUM) neurones, a group of cells located on the dorsal midline of each ganglion. We were interested whether and how these neurones are activated during walking and chose the semi-intact walking preparation of stick insects that offers to investigate single leg-stepping movements. DUM neurones were characterized in the thoracic nerve cord by backfilling lateral nerves. These backfills revealed a population of 6-8 efferent DUM cells per thoracic segment. Mesothoracic DUM cells were subsequently recorded during middle leg stepping and characterized by intracellular staining. Seven out of eight identified individual different types of DUM neurones were efferent. Seven types except the DUMna nl2 were tonically depolarized during middle leg stepping and additional phasic depolarizations in membrane potential linked to the stance phase of the middle leg were observed. These DUM neurones were all multimodal and received depolarizing synaptic drive when the abdomen, antennae or different parts of the leg were mechanically stimulated. We never observed hyperpolarising synaptic inputs to DUM neurones. Only one type of DUM neurone, DUMna, exhibited spontaneous rhythmic activity and was unaffected by different stimuli or walking movements.     

Uniparental loss of ribosomal DNA in the allotetraploid grass Zingeria trichopoda (2n = 8).

Analysis of the grass Zingeria trichopoda (2n = 8, 2C = 5.3 pg) revealed a dynamic evolution with the following characteristics. (i) Genomic in situ hybridization (GISH) demonstrates that Z. trichopoda evolved from an interspecific hybrid involving a species like contemporary Zingeria biebersteiniana (2n = 4) and a second species with a similar low number of chromosomes. The nucleus of Z. trichopoda is spatially organized at the genome level and the two parental genomes occupy distinct and separate domains of lateral arrangements. (ii) The copy number of the Z. biebersteiniana specific pericentromeric tandem repeat family Zbcen1 is drastically reduced in Z. trichopoda. (iii) GISH in combination with labeled rDNA sequences simultaneously discriminated the two parental genomes and the corresponding 5S and 45S rDNA sites. Hence, following allopolyploidization of Z. trichopoda the Z. biebersteiniana like parental chromosomes probably underwent drastic loss of 45S rDNA. This could have arisen either through the loss of Z. biebersteiniana derived 45S rDNA or through Z. trichopoda genome-wide homogenization of Z. biebersteiniana type 45S rDNA and subsequent elimination of 45S rDNA loci from Z. biebersteiniana derived chromosomes. Finally, 5S rDNA loci are present in both subgenomes of Z. trichopoda and the chromosomal position of these loci is similar for both Z. biebersteiniana and the Z. biebersteiniana like parental genome of Z. trichopoda.