Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Nested distributions of bat flies (Diptera: Streblidae) on Neotropical bats: artifact and specificity in host-parasite studies

We examined the structure of ectoparasitic bat fly infestations on 31 well‐sampled bat species, representing 4 Neotropical families. Sample sizes varied from 22 to 1057 bats per species, and bat species were infested by 4 to 27 bat fly species. Individual bats supported smaller infracommunities (the set of parasites co‐occurring on an individual host), ranging from 1 to 5 fly species in size, and no bat species had more than 6 bat fly species characteristically associated with it (its primary fly species). Nestedness analyses used system temperature (BINMATNEST algorithm) because it is particularly well‐suited for analysis of interaction networks, where parasite records may be nested among hosts and host individuals simultaneously nested among parasites. Most species exhibited very low system temperatures (mean 3.14°; range 0.14–12.28°). Simulations showed that nested structure for all 31 species was significantly stronger than simulated values under 2 of the 3 null hypotheses, and about half the species were also nested under the more stringent conditions of the third null hypothesis. Yet this structure disappears when analyses are restricted to “primary” associations of fly species (flies on their customary host species), which exclude records thought to be atypical, transient, or potential contaminants. Despite comprising a small fraction of total parasite records, such anomalies represent a considerable part of the statistical state‐space, offering the illusion of significant ecological structure. Only well understood and well documented systems can make distinctions between primary and other occurrence records. Generally, nestedness appears best developed in host‐parasite systems where infestations are long‐term and accumulate over time. Dynamic, short‐term infestations by highly mobile parasites like bat flies may appear to be nested, but such structure is better understood in terms of host specificity and accidental occurrences than in terms of prevalence, persistence, or hierarchical niche relations of the flies.     

Evolution after gene duplication: models, mechanisms, sequences, systems, and organisms

Gene duplication is postulated to have played a major role in the evolution of biological novelty. Here, gene duplication is examined across levels of biological organization in an attempt to create a unified picture of the mechanistic process by which gene duplication can have played a role in generating biodiversity. Neofunctionalization and subfunctionalization have been proposed as important processes driving the retention of duplicate genes. These models have foundations in population genetic theory, which is now being refined by explicit consideration of the structural constraints placed upon genes encoding proteins through physical chemistry. Further, such models can be examined in the context of comparative genomics, where an integration of gene-level evolution and species-level evolution allows an assessment of the frequency of duplication and the fate of duplicate genes. This process, of course, is dependent upon the biochemical role that duplicated genes play in biological systems, which is in turn dependent upon the mechanism of duplication: whole genome duplication involving a co-duplication of interacting partners vs. single gene duplication. Lastly, the role that these processes may have played in driving speciation is examined.     

Chiroptical properties of diamino carboxylic acids

Diamino carboxylic acids have recently come to the attention of scientists working in the field of early life and its development. These are the monomers of a hypothetic early form of genetic material, the so-called Peptide Nucleic Acid (PNA) (Nielson et al., Proc Natl Acad Sci USA 2000;97:3868-3871). Since all biopolymers rely on a specific handedness of their building blocks, the question of symmetry breaking occurs in diamino acids and PNA in the same way as in amino acids and proteins. One possible mechanism for triggering this, is asymmetric photochemistry in interstellar/circumstellar matter by means of circularly polarized light (Bailey et al., Science 2005;281:672-674; Bailey, Orig Life Evol Biosphere 2001;21:167-183; Buscherm?hle, Astrophys J 2005;624:821-826; Meierhenrich, Angew Chem Int Ed Engl 2005;44:5630-5634). Here we have measured the CD-spectra of four chiral diamino carboxylic acids, three of which were found in the Murchison meteorite (Meierhenrich, Proc Natl Acad Sci USA 2004;101:9182-9186). The spectra show a uniform peak at 200 nm. These results and additional quantum mechanical calculations of the involved molecular orbitals support the assumption that the process of symmetry breaking in diamino acids does not depend significantly on the length of the side chain. This means that one process alone could suffice to lead to symmetry breaking in all four measured diamino carboxylic acids and might even to some extent be transferable to monoamino acids, the monomers of proteins.     

Autophagic cell death induced by TrkA receptor activation in human glioblastoma cells

The neurotrophin receptor tropomyosin-related kinase A (TrkA) and its ligand nerve growth factor (NGF) are expressed in astrocytomas, and an inverse association of TrkA expression with malignancy grade was described. We hypothesized that TrkA expression might confer a growth disadvantage to glioblastoma cells. To analyze TrkA function and signaling, we transfected human TrkA cDNA into the human glioblastoma cell line G55. We obtained three stable clones, all of which responded with striking cytoplasmic vacuolation and subsequent cell death to NGF. Analyzing the mechanism of cell death, we could exclude apoptosis and cellular senescence. Instead, we identified several indications of autophagy: electron microscopy showed typical autophagic vacuoles; acridine orange staining revealed acidic vesicular organelles; acidification of acidic vesicular organelles was prevented using bafilomycin A1; cells displayed arrest in G2/M; increased processing of LC3 occurred; vacuolation was prevented by the autophagy inhibitor 3-methyladenine; no caspase activation was detected. We further found that both activation of ERK and c-Jun N-terminal kinase but not p38 were involved in autophagic vacuolation. To conclude, we identified autophagy as a novel mechanism of NGF-induced cell death. Our findings suggest that TrkA activation in human glioblastomas might be beneficial therapeutically, especially as several of the currently used chemotherapeutics also induce autophagic cell death.     

Effect of salinity changes on the bacterial diversity, photosynthesis and oxygen consumption of cyanobacterial mats from an intertidal flat of the Arabian Gulf

The effects of salinity fluctuation on bacterial diversity, rates of gross photosynthesis (GP) and oxygen consumption in the light (OCL) and in the dark (OCD) were investigated in three submerged cyanobacterial mats from a transect on an intertidal flat. The transect ran 1 km inland from the low water mark along an increasingly extreme habitat with respect to salinity. The response of GP, OCL and OCD in each sample to various salinities (65 per thousand, 100 per thousand, 150 per thousand and 200 per thousand) were compared. The obtained sequences and the number of unique operational taxonomic units showed clear differences in the mats' bacterial composition. While cyanobacteria decreased from the lower to the upper tidal mat, other bacterial groups such as Chloroflexus and Cytophaga/Flavobacteria/Bacteriodetes showed an opposite pattern with the highest dominance in the middle and upper tidal mats respectively. Gross photosynthesis and OCL at the ambient salinities of the mats decreased from the lower to the upper tidal zone. All mats, regardless of their tidal location, exhibited a decrease in areal GP, OCL and OCD rates at salinities > 100 per thousand. The extent of inhibition of these processes at higher salinities suggests an increase in salt adaptation of the mats microorganisms with distance from the low water line. We conclude that the resilience of microbial mats towards different salinity regimes on intertidal flats is accompanied by adjustment of the diversity and function of their microbial communities.     

Responses of Antarctic Iridaea cordata (Rhodophyta) tetraspores exposed to ultraviolet radiation

In Antarctica ozone depletion is highest during spring, coinciding with the reproduction of many seaweed species. Propagules are the life-stage of an alga most susceptible to environmental perturbations. Therefore, fertile thalli of Iridaea cordata (Turner) Bory (Rhodophyta) were collected in the eulittoral of King George Island (Antarctica) to examine spore susceptibility to ultraviolet radiation (UVR). In the laboratory, freshly released tetraspores were exposed to photosynthetically active radiation (PAR) (400–700 nm), PAR+UV-A (320–700 nm) or PAR+UV-A+UV-B (280–700 nm). Photosynthetic efficiency was measured during 1–8 h of exposure and after 48 h of recovery. Additionally, mycosporine-like amino acids (MAAs) and DNA damage were determined. Saturating irradiance of photosynthesis of freshly released tetraspores was 57 µmol photons m⁻² s⁻¹. Exposure to increasing fluence of PAR reduced photosynthetic efficiency. UVR further decreased the photosynthetic efficiencies of the tetraspores but spores were able to recover completely after UVR exposure and 2 days post-cultivation under low PAR. DNA damage was minimal and lesions were effectively repaired under photoreactivating light. Concentrations of the MAAs shinorine and palythine were higher in tetraspores treated with UVR than in spores only exposed to PAR. Generally, the tetraspores show a good UV tolerance. This flexible response of the tetraspores of this species to changing radiation conditions enables the alga to grow along a considerable depth gradient from the sublittoral to the eulittoral where they can be exposed to enhanced UVBR under conditions of stratospheric ozone depletion.     

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome‐wide association study

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome‐wide association study in an F₂ Charolais × German Holstein cross‐breed population to identify genetic variants that affect behaviour‐related traits assessed in an open‐field and novel‐object test and analysed their putative impact on milk performance. Of 37 201 tested single nucleotide polymorphism (SNPs), four showed a genome‐wide and 37 a chromosome‐wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel‐object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.     

Analysis of Genes Involved in Body Weight Regulation by Targeted Re-Sequencing

Introduction

Genes involved in body weight regulation that were previously investigated in genome-wide association studies (GWAS) and in animal models were target-enriched followed by massive parallel next generation sequencing.

Methods

We enriched and re-sequenced continuous genomic regions comprising FTO, MC4R, TMEM18, SDCCAG8, TKNS, MSRA and TBC1D1 in a screening sample of 196 extremely obese children and adolescents with age and sex specific body mass index (BMI) ≥ 99^th percentile and 176 lean adults (BMI ≤ 15^th percentile). 22 variants were confirmed by Sanger sequencing. Genotyping was performed in up to 705 independent obesity trios (extremely obese child and both parents), 243 extremely obese cases and 261 lean adults.

Results and Conclusion

We detected 20 different non-synonymous variants, one frame shift and one nonsense mutation in the 7 continuous genomic regions in study groups of different weight extremes. For SNP Arg695Cys (rs58983546) in TBC1D1 we detected nominal association with obesity (p_TDT = 0.03 in 705 trios). Eleven of the variants were rare, thus were only detected heterozygously in up to ten individual(s) of the complete screening sample of 372 individuals. Two of them (in FTO and MSRA) were found in lean individuals, nine in extremely obese. In silico analyses of the 11 variants did not reveal functional implications for the mutations. Concordant with our hypothesis we detected a rare variant that potentially leads to loss of FTO function in a lean individual. For TBC1D1, in contrary to our hypothesis, the loss of function variant (Arg443Stop) was found in an obese individual. Functional in vitro studies are warranted.     

Identification of specific miRNAs targeting proteins of the apical junctional complex that simulate the probiotic effect of E. coli Nissle 1917 on T84 epithelial cells

In the intestine, dysregulation of miRNA is associated with inflammation, disruption of the gastrointestinal barrier, and the onset of gastrointestinal disorders. This study identifies miRNAs involved in the maintenance of intercellular junctions and barrier integrity. For the functional identification of barrier affecting miRNAs, we took advantage of the barrier-enforcing effects of the probiotic bacterium Escherichia coli Nissle 1917 (EcN) which can be monitored by enhanced transepithelial resistance (TER). miRNA-profiling of T84 monolayers prior and after co-incubation with EcN revealed for the first time differentially regulated miRNAs (miR-203, miR-483-3p, miR-595) targeting tight junction (TJ) proteins. Using real-time PCR, Western blotting and specific miRNA mimics, we showed that these miRNAs are involved in the regulation of barrier function by modulating the expression of regulatory and structural components of tight junctional complexes. Furthermore, specific inhibitors directed at these miRNA abrogated the disturbance of tight junctions induced by enteropathogenic E. coli (EPEC). The half-maximal inhibitory concentration (IC(50)) was determined to 340 nM by monitoring inhibitor kinetics. In summary, we conclude that specific miRNAs effect regulatory as well as structural proteins of the junctional complex which in turn are involved in the barrier enhancing effect of EcN. Hence, we suggest that the application of miRNAs might be refined and further developed as a novel supportive strategy for the treatment of gastrointestinal disorders.