Search for informative polymorphisms in candidate genes: clock genes and circadian behaviour in blue tits

The identification of functional polymorphisms in genes that underlie behavioural trait variation is a challenging but intriguing task in evolutionary biology. Given the wealth of genomic data and the increasing number of genotype–phenotype association studies in model organisms, one can ask whether and how this information can be used for non-model organisms. Here we describe two strategies to search for likely functional polymorphisms in candidate genes in a bird species that has been intensively studied by behavioural and population ecologists, the blue tit Cyanistes caeruleus. In the first approach we searched for repeating elements in coding regions of the genome using information about repeats in Gallus gallus genes. The rationale is that tandem-repeat elements have a high potential to be polymorphic and functional. The second strategy aimed to replicate reported genotype–phenotype association studies by extrapolating results from model organisms to our study species. Both strategies showed high success rates with respect to finding homologous gene regions and potentially informative genetic variants in the genes AANAT, ADCYAP1, CKIε, CLOCK, CREB1, NPAS2 and PERIOD2. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Altered stress stimulation of inward rectifier potassium channels in Andersen-Tawil syndrome

Inward rectifier potassium channels of the Kir2 subfamily are important determinants of the electrical activity of brain and muscle cells. Genetic mutations in Kir2.1 associate with Andersen-Tawil syndrome (ATS), a familial disorder leading to stress-triggered periodic paralysis and ventricular arrhythmia. To identify the molecular mechanisms of this stress trigger, we analyze Kir channel function and localization electrophysiologically and by time-resolved confocal microscopy. Furthermore, we employ a mathematical model of muscular membrane potential. We identify a novel corticoid signaling pathway that, when activated by glucocorticoids, leads to enrichment of Kir2 channels in the plasma membranes of mammalian cell lines and isolated cardiac and skeletal muscle cells. We further demonstrate that activation of this pathway can either partly restore (40% of cases) or further impair (20% of cases) the function of mutant ATS channels, depending on the particular Kir2.1 mutation. This means that glucocorticoid treatment might either alleviate or deteriorate symptoms of ATS depending on the patient's individual Kir2.1 genotype. Thus, our findings provide a possible explanation for the contradictory effects of glucocorticoid treatment on symptoms in patients with ATS and may open new pathways for the design of personalized medicines in ATS therapy.     

Interocular yoking in human saccades examined by mutual information analysis

Background

Saccadic eye movements align the two eyes precisely to foveate a target. Trial-by-trial variance of eye movement is always observed within an identical experimental condition. This has often been treated as experimental error without addressing its significance. The present study examined statistical linkages between the two eyes’ movements, namely interocular yoking, for the variance of eye position and velocity.

Methods

Horizontal saccadic movements were recorded from twelve right-eye-dominant subjects while they decided on saccade direction in Go-Only sessions and on both saccade execution and direction in Go/NoGo sessions. We used infrared corneal reflection to record simultaneously and independently the movement of each eye. Quantitative measures of yoking were provided by mutual information analysis of eye position or velocity, which is sensitive to both linear and non-linear relationships between the eyes’ movements. Our mutual information analysis relied on the variance of the eyes movements in each experimental condition. The range of movements for each eye varies for different conditions so yoking was further studied by comparing GO-Only vs. Go/NoGo sessions, leftward vs. rightward saccades.

Results

Mutual information analysis showed that velocity yoking preceded positional yoking. Cognitive load increased trial variances of velocity with no increase in velocity yoking, suggesting that cognitive load may alter neural processes in areas to which oculomotor control is not tightly linked. The comparison between experimental conditions showed that interocular linkage in velocity variance of the right eye lagged that of the left eye during saccades.

Conclusions

We conclude quantitative measure of interocular yoking based on trial-to-trial variance within a condition, as well as variance between conditions, provides a powerful tool for studying the binocular movement mechanism.

     

Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody

Recombinant protein therapeutics have become increasingly useful in combating human diseases, such as cancer and those of genetic origin. One quality concern for protein therapeutics is the content and the structure of the aggregated proteins in the product, due to the potential immunogenicity of these aggregates. Collective efforts have led to a better understanding of some types of protein aggregates, and have revealed the diversity in the structure and cause of protein aggregation. In this work we used a broad range of analytical techniques to characterize the quinary structure (complexes in which each composing unit maintains native quaternary structure) of the stable non-covalent dimer and oligomers of a monoclonal IgG1λ antibody. The results supported a mechanism of intermolecular domain exchange involving the Fab domains of 2 or more IgG molecules. This mechanism can account for the native-like higher order (secondary, tertiary and disulfide bonding) structure, the stability of the non-covalent multimers, and the previously observed partial loss of the antigen-binding sites without changing the antigen-binding affinity and kinetics of the remaining sites (Luo et al., 2009, mAbs 1:491). Furthermore, the previously observed increase in the apparent affinity to various Fcγ receptors (ibid), which may potentially promote immunogenicity, was also explained by the quinary structure proposed here. Several lines of evidence indicated that the formation of multimers by the mechanism of intermolecular domain exchange took place mostly during expression, not in the purified materials. The findings in this work will advance our knowledge of the mechanisms for aggregation in therapeutic monoclonal antibodies.     

Optimization of an osmotic shock procedure for isolation of a protein product expressed in E. coli

Small-scale experiments were performed to evaluate the impact of several processing variables on recovery by osmotic shock of a protein product expressed in E. coli utilizing feed material manufactured at large scale. Equilibration time, type and concentration of osmotic supplements, and pH of the osmotic shock buffer were found to have significant impact on the osmotic shock recovery. Improved recovery was documented under conditions of increased equilibration time before the osmotic shock and increased Tris-HCl concentration in the osmotic shock buffer, as well as initial dispersal of cells into the osmotic shock buffer. Recovery of up to 65% was achieved under the optimized conditions.     

Antagonistic action of a 25-carboxylic ester analogue of 1alpha, 25-dihydroxyvitamin D3 is mediated by a lack of ligand-induced vitamin D receptor interaction with coactivators

A 25-carboxylic ester analogue of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)), ZK159222, was described as a novel type of antagonist of 1alpha,25-(OH)(2)D(3) signaling. The ligand sensitivity of ZK159222, in facilitating complex formation between 1alpha,25-(OH)(2)D(3) receptor (VDR) and the retinoid X receptor (RXR) on a 1alpha,25-(OH)(2)D(3) response element (VDRE), was approximately 7-fold lower when compared with 1alpha,25-(OH)(2)D(3). However, ZK159222 was not able to promote a ligand-dependent interaction of the VDR with the coactivator proteins SRC-1, TIF2, and RAC3, neither in solution nor in a complex with RXR on DNA. Functional analysis in HeLa and COS-7 cells demonstrated a 10-100-fold lower ligand sensitivity for ZK159222 than for 1alpha, 25-(OH)(2)D(3) and, most interestingly, a potency that was drastically reduced compared with 1alpha,25-(OH)(2)D(3). A cotreatment of 1alpha,25-(OH)(2)D(3) with a 100-fold higher concentration of ZK159222 resulted in a prominent antagonistic effect both in functional in vivo and in in vitro assays. These data suggest that the antagonistic action of ZK159222 is due to a lack of ligand-induced interaction of the VDR with coactivators with a parallel ligand sensitivity, which is sufficient for competition with the natural hormone for VDR binding.     

Articular Joint Lubricants during Osteoarthritis and Rheumatoid Arthritis Display Altered Levels and Molecular Species

Background

Hyaluronic acid (HA), lubricin, and phospholipid species (PLs) contribute independently or together to the boundary lubrication of articular joints that is provided by synovial fluid (SF). Our study is the first reporting quantitative data about the molecular weight (MW) forms of HA, lubricin, and PLs in SF from cohorts of healthy donors, patients with early (eOA)- or late (lOA)-stage osteoarthritis (OA), and patients with active rheumatoid arthritis (RA).

Methods

We used human SF from unaffected controls, eOA, lOA, and RA. HA and lubricin levels were measured by enzyme-linked immunosorbent assay. PLs was quantified by electrospray ionization tandem mass spectrometry. Fatty acids (FAs) were analyzed by gas chromatography, coupled with mass spectrometry. The MW distribution of HA was determined by agarose gel electrophoresis.

Results

Compared with control SF, the concentrations of HA and lubricin were lower in OA and RA SF, whereas those of PLs were higher in OA and RA SF. Moreover, the MW distribution of HA shifted toward the lower ranges in OA and RA SF. We noted distinct alterations between cohorts in the relative distribution of PLs and the degree of FA saturation and chain lengths of FAs.

Conclusions

The levels, composition, and MW distribution of all currently known lubricants in SF—HA, lubricin, PLs—vary with joint disease and stage of OA. Our study is the first delivering a comprehensive view about all joint lubricants during health and widespread joint diseases. Thus, we provide the framework to develop new optimal compounded lubricants to reduce joint destruction.     

KCO1 is a component of the slow-vacuolar (SV) ion channel

The Arabidopsis double pore K+ channel KCO1 was fused to green fluorescent protein and expressed in tobacco protoplasts. Microscopic analysis revealed a bright green fluorescence at the vacuolar membrane. RT-PCR experiments showed that KCO1 is expressed in the mesophyll. Vacuoles from Arabidopsis wild-type and kco1 knockout plants were isolated for patch-clamp analyses. Currents mediated by slow-activating vacuolar (SV) channels of mesophyll cell vacuoles were significantly smaller in kco1 plants compared to the wild-type. This shows that KCO1 is involved in the formation of SV channels.     

Spectrum of mutations in the major human skeletal muscle chloride channel gene (CLCN1) leading to myotonia.

Autosomal dominant myotonia congenita and autosomal recessive generalized myotonia (GM) are genetic disorders characterized by the symptom of myotonia, which is based on an electrical instability of the muscle fiber membrane. Recently, these two phenotypes have been associated with mutations in the major muscle chloride channel gene CLCN1 on human chromosome 7q35. We have systematically screened the open reading frame of the CLCN1 gene for mutations by SSC analysis (SSCA) in a panel of 24 families and 17 single unrelated patients with human myotonia. By direct sequencing of aberrant SSCA conformers were revealed 15 different mutations in a total of 18 unrelated families and 13 single patients. Of these, 10 were novel (7 missense mutations, 2 mutations leading to frameshift, and 1 mutation predicted to affect normal splicing). In our overall sample of 94 GM chromosomes we were able to detect 48 (51%) mutant GM alleles. Three mutations (F413C), R894X, and a 14-bp deletion in exon 13) account for 32% of the GM chromosomes in the German population. Our finding that A437T is probably a polymorphism is in contrast to a recent report that the recessive phenotype GM is associated with this amino acid change. We also demonstrate that the R894X mutation may act as a recessive or a dominant mutation in the CLCN1 gene, probably depending on the genetic background. Functional expression of the R894X mutant in Xenopus oocytes revealed a large reduction, but not complete abolition, of chloride currents. Further, it had a weak dominant negative effect on wild-type currents in coexpression studies. Reduction of currents predicted for heterozygous carriers are close to the borderline value, which is sufficient to elicit myotonia.