Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex. Offprint requests to: W. Hanke     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.     

Binding of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate opens the pathway for protons through the chloroplast ATPase complex

The effect of 2'(3')-O-(2,4,6-trinitrophenyl)-adenosine 5'-diphosphate (TNP-ADP) on photophosphorylation and on the proton conductivity of the thylakoid membrane has been investigated. The results show that TNP-ADP is a potent competitive inhibitor of photophosphorylation (Ki = 1-2 microM). Moreover, in the absence of ADP and Pi, TNP-ADP accelerates basal electron transport of chloroplasts. Addition of ADP, which promotes release of the analogue from CF1, completely reverses this effect of TNP-ADP; likewise Pi alone reverses stimulation of electron transport by TNP-ADP. Dicyclohexylcarbodiimide treatment, which is known to close CF0 to H+, completely abolishes the effect of TNP-ADP. The measurements of the alkalization of the medium and the acidification of the thylakoid lumen following single turnover flashes showed that binding of TNP-ADP to CF1 increased membrane permeability for H+. Further results suggest that binding of TNP-ADP to the catalytic site of CF1 opens the CF0-CF1 complex for H+. Since ADP, as well as Pi alone, reverses the effect, it is concluded that TNP-ADP induces a conformation of the CF0-CF1 complex similar to the one triggered by simultaneous binding of ADP plus Pi. This may be achieved by interaction of the TNP residue with the Pi binding site. Thus it seems that the status of the catalytic site(s) in CF1 can be transmitted to the CF0 part to control proton flux through the ATPase complex in an economically reasonable way.     

Evidence for diet partitioning among three coexisting native freshwater fishes in South Africa's Cape Fold Ecoregion

The partitioning of limited resources commonly explains how different species can coexist within the same ecological community. In this 2010 study, the diets of three coexisting freshwater fishes (Cape galaxias Galaxias zebratus, n = 27; Cape kurper Sandelia capensis, n = 60; Breede River redfin Pseudobarbus burchelli, n = 77) were characterised and compared in three headwater streams in South Africa's Cape Fold Ecoregion using gut contents and stable isotope analyses. These data were analysed to ascertain whether the three species exploit distinct trophic niches. Both approaches provided evidence that these species occupy different trophic niches, though with some overlap. However, dietary differences among sites were not consistent and were probably influenced by site-specific factors like resource availability. Pseudobarbus burchelli had a broader niche breadth at Tierkloof Stream than the other two species, but not at Waaihoek or Tierstel Streams. Our results also suggest that P. burchelli consumed a more omnivorous diet than do the other two species, whereas S. capensis occupied a higher trophic position than the other two species and consumed vertebrates. Our findings suggest that these species occupy non-equivalent feeding niches in Cape Fold Ecoregion headwater streams, and that diet partitioning might facilitate their coexistence in these systems.     

Total sleep deprivation does not significantly degrade semantic encoding

ABSTRACT

Sleep deprivation impairs performance on cognitive tasks, but it is unclear which cognitive processes it degrades. We administered a semantic matching task with variable stimulus onset asynchrony (SOA) and both speeded and self-paced trial blocks. The task was administered at the baseline and 24 hours later after 30.8 hours of total sleep deprivation (TSD) or matching well-rested control. After sleep deprivation, the 20% slowest response times (RTs) were significantly increased. However, the semantic encoding time component of the RTs remained at baseline level. Thus, the performance impairment induced by sleep deprivation on this task occurred in cognitive processes downstream of semantic encoding.     

Elevated levels of fibrinogen-derived endogenous citrullinated peptides in synovial fluid of rheumatoid arthritis patients

Introduction

Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammation of the joints and the presence of autoantibodies directed against proteins containing the non-standard arginine-derived amino acid citrulline. The protein fibrinogen, which has an essential role in blood clotting, is one of the most prominent citrullinated autoantigens in RA, particularly because it can be found in the inflamed tissue of affected joints. Here, we set out to analyze the presence of citrullinated endogenous peptides in the synovial fluid of RA and arthritic control patients.

Methods

Endogenous peptides were isolated from the synovial fluid of RA patients and controls by filtration and solid phase extraction. The peptides were identified and quantified using high-resolution liquid chromatography-mass spectrometry.

Results

Our data reveal that the synovial fluid of RA patients contains soluble endogenous peptides, derived from fibrinogen, containing significant amounts of citrulline residues and, in some cases, also phosphorylated serine. Several citrullinated peptides are found to be more abundantly present in the synovial fluid of RA patients compared to patients suffering from other inflammatory diseases affecting the joints.

Conclusions

The increased presence of citrullinated peptides in RA patients points toward a possible specific role of these peptides in the immune response at the basis of the recognition of citrullinated peptides and proteins by RA patient autoantibodies.     

Membrane localization diversity of TPK channels and their physiological role

Potassium (K) is one of the major nutrients that is essential for plant growth and development. The majority of cellular K⁺ resides in the vacuole and tonoplast K⁺ channels of the TPK (Two Pore K) family are main players in cellular K⁺ homeostasis. All TPK channels were previously reported to be expressed in the tonoplast of the large central lytic vacuole (LV) except for one isoform in Arabidopsis that resides in the plasma membrane. However, plant cells often contain more than one type of vacuole that coexist in the same cell. We recently showed that two TPK isoforms (OsTPKa and OsTPKb) from Oryza sativa localize to different vacuoles with OsTPKa predominantly found in the LV tonoplast and OsTPKb primarily in smaller compartments that resemble small vacuoles (SVs). Our study further revealed that it is the C-terminal domain that determines differential targeting of OsTPKa and OsTPKb. Three C-terminal amino acids were particularly relevant for targeting TPKs to their respective endomembranes. In this addendum we further evaluate how the different localization of TPKa and TPKb impact on their physiological role and how TPKs provide a potential tool to study the physiology of different types of vacuole.Key words: TPK channels, small vacuoles, vacuolar targeting, potassiumThe roles of plant vacuolar K⁺ channels are diverse and include potassium homeostasis, turgor regulation and responses to abiotic stress. Vacuolar K⁺-selective channels belong to two-pore K⁺ (TPK) channel families which have been found in genomes of many plant species such as Arabidopsis, poplar, Physcomitrella, Eucalyptus, barley, potato, rice and tobacco (Fig. 1). TPKs have structural similarity to mammalian “tandem P domain” channels with a secondary structure that contains four transmembrane domains and two pore regions (Fig. 2).^1–5 TPK channels have pore regions with a GYGD signature that endows K⁺ selectivity and a variable number of Ca²⁺ binding EF domains in the C terminus.^3–8 One of the best characterized members of the TPK family is AtTPK1 from Arabidopsis thaliana. AtTPK1 activity is voltage independent but sensitive to cytosolic Ca²⁺, cytosolic pH and N-terminal phosphorylation by 14-3-3 proteins.^5,6,8,9 In Arabidopsis, AtTPK1 expresses in the large lytic vacuole (LV) and plays roles in cellular K⁺ homeostasis, K⁺-release during stomatal closure and seed germination.^4,5 Other members of the Arabidopsis TPK family (AtTPK2, AtTPK3, AtTPK5) have been shown to localize to the LV but also showed some expression in smaller, vesicle-like, compartments.⁴ However, none of these isoforms appears to form functional channels in planta although our experiments with heterologous expression of AtTPK3 and AtTPK5 in the K⁺ uptake deficient E. coli LB2003 demonstrates complementation of bacterial growth phenotype (Isayenkov S, et al. unpublished results). Equally intriguing, is the plasma membrane localization of the Arabidopsis TPK4 isoform, in spite of its sequence being very similar to that of other TPKs.¹⁰Open in a separate windowFigure 1Phylogenetic tree of plant TPKs. The three main clusters of TPKs comprise: Cluster 1 with AtTPK1-like channels; Cluster 2 with AtTPK3/TPK5-like channels; Cluster 3 with barley HvTPKb. Bootstrap analysis was performed using ‘Molecular Evolutionary Genetics Analysis, MEGA4’ software available at www.megasoftware.net/mega4/megaOpen in a separate windowFigure 2Two-pore potassium channel secondary structure. TPK channels comprise four transmembrane domains (1–4) and two pore regions (P) per subunit. Functional channels are formed from two subunits. In most TPKs, both P regions contain a K⁺ selectivity signature, GYGD. However, the tobacco NtTPKa isoform has different motifs in the second P domain. In the N terminal region, TPKs have a 14-3-3 binding domain that impact on channel activity, with the binding of 14-3-3 protein leading to channel activation. C-termini of TPKs show a varying number of putative Ca²⁺ binding “EF hands” which may vary from zero to two.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30?kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.