Characterization of the two coactivator-interacting surfaces of the androgen receptor and their relative role in transcriptional control

The androgen receptor interacts with the p160 coactivators via two surfaces, one in the ligand binding domain and one in the amino-terminal domain. The ligand binding domain interacts with the nuclear receptor signature motifs, whereas the amino-terminal domain has a high affinity for a specific glutamine-rich region in the p160s. We here describe the implication of two conserved motifs in the latter interaction. The amino-terminal domain of the androgen receptor is a very strong activation domain constituent of Tau5, which is mainly active in the absence of the ligand binding domain, and Tau1, which is only active in the presence of the ligand binding domain. Both domains are, however, implicated in the recruitment of the p160s. Mutation analysis of the p160s has shown that the relative contribution of the two recruitment mechanisms via the signature motifs or via the glutamine-rich region depend on the nature of the enhancers tested. We propose, therefore, that the androgen receptor-coactivator complex has several alternative conformations, depending partially on the context of the enhancer.     

Influence of Water Content and Temperature on Molecular Mobility and Intracellular Glasses in Seeds and Pollen

Although the occurrence of intracellular glasses in seeds and pollen has been established, physical properties such as rotational correlation times and viscosity have not been studied extensively. Using electron paramagnetic resonance spectroscopy, we examined changes in the molecular mobility of the hydrophilic nitroxide spin probe 3-carboxy-proxyl during melting of intracellular glasses in axes of pea (Pisum sativum L.) seeds and cattail (Typha latifolia L.) pollen. The rotational correlation time of the spin probe in intracellular glasses of both organisms was approximately 10⁻³ s. Using the distance between the outer extrema of the electron paramagnetic resonance spectrum (2A_zz) as a measure of molecular mobility, we found a sharp increase in mobility at a definite temperature during heating. This temperature increased with decreasing water content of the samples. Differential scanning calorimetry data on these samples indicated that this sharp increase corresponded to melting of the glassy matrix. Molecular mobility was found to be inversely correlated with storage stability. With decreasing water content, the molecular mobility reached a minimum, and increased again at very low water content. Minimum mobility and maximum storage stability occurred at a similar water content. This correlation suggests that storage stability might be at least partially controlled by molecular mobility. At low temperatures, when storage longevity cannot be determined on a realistic time scale, 2A_zz measurements can provide an estimate of the optimum storage conditions.     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.     

Quantifying the Displacement of Mismatches in Multiple Sequence Alignment Benchmarks

Multiple Sequence Alignment (MSA) methods are typically benchmarked on sets of reference alignments. The quality of the alignment can then be represented by the sum-of-pairs (SP) or column (CS) scores, which measure the agreement between a reference and corresponding query alignment. Both the SP and CS scores treat mismatches between a query and reference alignment as equally bad, and do not take the separation into account between two amino acids in the query alignment, that should have been matched according to the reference alignment. This is significant since the magnitude of alignment shifts is often of relevance in biological analyses, including homology modeling and MSA refinement/manual alignment editing. In this study we develop a new alignment benchmark scoring scheme, SPdist, that takes the degree of discordance of mismatches into account by measuring the sequence distance between mismatched residue pairs in the query alignment. Using this new score along with the standard SP score, we investigate the discriminatory behavior of the new score by assessing how well six different MSA methods perform with respect to BAliBASE reference alignments. The SP score and the SPdist score yield very similar outcomes when the reference and query alignments are close. However, for more divergent reference alignments the SPdist score is able to distinguish between methods that keep alignments approximately close to the reference and those exhibiting larger shifts. We observed that by using SPdist together with SP scoring we were able to better delineate the alignment quality difference between alternative MSA methods. With a case study we exemplify why it is important, from a biological perspective, to consider the separation of mismatches. The SPdist scoring scheme has been implemented in the VerAlign web server (http://www.ibi.vu.nl/programs/veralignwww/). The code for calculating SPdist score is also available upon request.     

Insights in KIR2.1 channel structure and function by an evolutionary approach; cloning and functional characterization of the first reptilian inward rectifier channel KIR2.1, derived from the California kingsnake (Lampropeltis getula californiae)

Potassium inward rectifier K_IR2.1 channels contribute to the stable resting membrane potential in a variety of muscle and neuronal cell-types. Mutations in the K_IR2.1 gene KCNJ2 have been associated with human disease, such as cardiac arrhythmias and periodic paralysis. Crystal structure and homology modelling of K_IR2.1 channels combined with functional current measurements provided valuable insights in mechanisms underlying channel function. K_IR2.1 channels have been cloned and analyzed from all main vertebrate phyla, except reptilians. To address this lacuna, we set out to clone reptilian K_IR2.1 channels. Using a degenerated primer set we cloned the KCNJ2 coding regions from muscle tissue of turtle, snake, bear, quail and bream, and compared their deduced amino acid sequences with those of K_IR2.1 sequences from 26 different animal species obtained from Genbank. Furthermore, expression constructs were prepared for functional electrophysiological studies of ectopically expressed K_IR2.1 ion channels. In general, KCNJ2 gene evolution followed normal phylogenetic patterns, however turtle K_IR2.1 ion channel sequence is more homologues to avians than to snake. Alignment of all 31 K_IR2.1 sequences showed that all disease causing K_IR2.1 mutations, except V93I, V123G and N318S, are fully conserved. Homology models were built to provide structural insights into species specific amino acid substitutions. Snake K_IR2.1 channels became expressed at the plasmamembrane and produced typical barium sensitive (IC₅₀ ∼6 μM) inward rectifier currents.     

Longitudinal assessment of the effect of concentration on stream N uptake rates in an urbanizing watershed

We examined the effect of concentration on nitrogen uptake patterns for a suburban stream in Maryland and addressed the question: How does NO₃ ^? uptake change as a function of concentration and how do uptake patterns compare with those found for NH₄ ⁺? We applied a longitudinal (stream channel corridor) approach in a forested stream section and conducted short-term nutrient addition experiments in late summer 2004. In the downstream direction, NO₃ ^? concentrations decreased because of residential development in headwaters and downstream dilution; NH₄ ⁺ concentrations slightly increased. The uptake patterns for NO₃ ^? were very different from NH₄ ⁺. While NH₄ ⁺ had a typical negative relationship between first-order uptake rate constant (K _c ) and stream size, NO₃ ^? had a reverse pattern. We found differences for other metrics, including uptake velocity (V _f ) and areal uptake rate (U). We attributed these differences to a stream size effect, a concentration effect and a biological uptake capacity effect. For NO₃ ^? these combined effects produced a downstream increase in K _c , V _f and U; for NH₄ ⁺ they produced a downstream decrease in K _c and V _f , and a not well defined pattern for U. We attributed a downstream increase in NO₃ ^? uptake capacity to an increase in hyporheic exchange and a likely increase in carbon availability. We also found that K _c and V _f were indirectly related with concentration. Similar evidence of ‘nutrient saturation’ has been reported in other recent studies. Our results suggest that higher-order uptake models might be warranted when scaling NO₃ ^? uptake across watersheds that are subject to increased nitrogen loading.     

Longitudinal and seasonal variation of stream N uptake in an urbanizing watershed: effect of organic matter, stream size, transient storage and debris dams

We examined seasonal and spatial linkages between N cycling and organic matter for a suburban stream in Maryland and addressed the question: How do longitudinal NH₄ ⁺ uptake patterns vary seasonally and what is the effect of organic matter, stream size, transient storage and debris dams? We applied a longitudinal (stream channel corridor) approach in a forested stream section and conducted short-term nutrient addition experiments (adapted to account for the effect of nutrient saturation) covering 14–16 reaches, and compared two distinct seasons (late fall 2003 and late summer 2004). Longitudinal NH₄ ⁺ uptake rate patterns had a distinct seasonal reversal; fall had the highest uptake rates in the upper reaches, while summer had the highest uptake rates in the lower reaches. This seasonal reversal was attributed to organic matter and evidenced by DON patterns. Transient storage did not have an expected effect on uptake rates in fall because it was confounded by leaf litter; litter produced higher uptakes, but also may have reduced transient storage. In summer however, uptake rates had a positive correlation with transient storage. Debris dams had no distinct effect on uptake in fall because of their recent formation. In summer however, the debris dam effect was significant; although the debris dams were hydraulically inactive then, the upstream reaches had 2–5 fold higher uptake rates. The seasonal and longitudinal differences in NH₄ ⁺ uptake reflect interactions between flow conditions and the role of organic matter. Urbanization can alter both of these characteristics, hence affect stream N processing.     

Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin.