Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8–87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.     

The effect of electrostatic charge of food particles on capture efficiency by Oxyrrhis marina Dujardin (dinoflagellate)

Laboratory experiments were carried out to investigate the effect of food quality, measured as surface charge of the particles, on capture efficiency and ingestion rate by the heterotrophic dinoflagellate Oxyrrhis marina. Fluorescent particles in two size classes of around 1 and 4 microm and of 7 different qualities were offered to the flagellate: carbohydrate and albumin particles, the algae Synechocystis spec. and Chlorella spec., carboxylated microspheres, silicate particles and bacteria. Rates of particle uptake showed significant differences depending on particle size and quality, and ranged from 0 to 4 particles cell(-1) h(-1). Ingestion rates were up to 4 times higher for 4 pm particles than for 1 microm particles, which indicates strong size-selective feeding. Our main result is that the surface charge or zeta potential, of artificial particles, i.e. carboxylated microspheres (> or = -107 mV) and silicate particles, strongly differ from more natural and natural food (< or = -17 mV). For both size classes Oxyrrhis had ingestion rates up to 4 times higher for particles with less negative charge, such as albumin particles or algae. Thus, the zeta potential of the model food should be considered in experimental design. Particles with a zeta potential similar to that of natural food, e.g. albumin, seem to be the preferred model food.     

Mitochondrial apoptosis induced by BH3‐only molecules in the exclusive presence of endoplasmic reticular Bak

Bak and Bax are critical apoptotic mediators that naturally localize to both mitochondria and the endoplasmic reticulum (ER). Although it is generally accepted that mitochondrial expression of Bak or Bax suffices for apoptosis initiated by BH3‐only homologues, it is currently unclear whether their reticular counterparts may have a similar potential. In this study, we show that cells exclusively expressing Bak in endoplasmic membranes undergo cytochrome c mobilization and mitochondrial apoptosis in response to BimEL and Puma, even when these BH3‐only molecules are also targeted to the ER. Surprisingly, calcium was necessary but not sufficient to drive the pathway, despite normal ER calcium levels. We provide evidence that calcium functions coordinately with the ER‐stress surveillance machinery IRE1α/TRAF2 to transmit apoptotic signals from the reticulum to mitochondria. These results indicate that BH3‐only mediators can rely on reticular Bak to activate an ER‐to‐mitochondria signalling route able to induce cytochrome c release and apoptosis independently of the canonical Bak,Bax‐dependent mitochondrial gateway, thus revealing a new layer of complexity in apoptotic regulation.     

Identification and characterization of NAGNAG alternative splicing in the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Background  

Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss.     

Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants

Parkinson's disease (PD)‐associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line‐derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin‐based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret^MEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret^MEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret‐mediated cell protection in a situation relevant for human PD.     

Description of Geodermatophilus amargosae sp. nov., to Accommodate the Not Validly Named Geodermatophilus obscurus subsp. amargosae (Luedemann, 1968)

A novel, gram reaction positive aerobic actinobacterium, designated G12^T, not validly named as Geodermatophilus obscurus subsp. amargosae, was accessed in the DSMZ open collection as DSM 46136^T. The optimal growth was at 2,535 °C, at pH 6.0–12.0 and in the absence of NaCl, forming greenish-black-coloured colonies on GYM agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G+C content of the strain was 73.0 mol%. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H₄) was the dominant menaquinone and galactose was detected as a diagnostic sugar. The major cellular fatty acid was branched-chain saturated acid iso-C_15:0. The 16S rRNA gene showed 94.2–99.5 % sequence identity with the members of the genus Geodermatophilus. Based on the chemotaxonomic results and 16S rRNA gene sequence analysis, strain G12^T is proposed to represent a novel species, Geodermatophilus amargosae. Type strain is G12^T [=G96] (=DSM 46136^T = CCUG 62971^T = MTCC 11559^T = ATCC 25081^T = JCM 3153^T = NBRC 13316^T = NRRL B-3578^T = KCTC 9360^T). The INSDC accession number is HF679056.     

Influence of local climate and climate change on aeroterrestrial phototrophic biofilms

Aeroterrestrial phototrophic biofilms colonize natural and man-made surfaces and may damage the material they settle on. The occurrence of biofilms varies between regions with different climatic conditions. The aim of this study was to evaluate the influence of meteorological factors on the growth of aeroterrestrial phototrophs. Phototrophic biomass was recorded on roof tiles at six sites within Germany five times over a period of five years and compared to climatic parameters from neighboring weather stations. All correlating meteorological factors influenced water availability on the surface of the roof tiles. The results indicate that the frequency of rainy days and not the mean precipitation per season is more important for biofilm proliferation. It is also inferred that the macroclimate is more important than the microclimate. In conclusion, changed (regional) climatic conditions may determine where in central Europe global change will promote or inhibit phototrophic growth in the future.