Evidence for DNA-binding domain--ligand-binding domain communications in the androgen receptor

DNA binding as well as ligand binding by nuclear receptors has been studied extensively. Both binding functions are attributed to isolated domains of which the structure is known. The crystal structure of a complete receptor in complex with its ligand and DNA-response element, however, has been solved only for the peroxisome proliferator-activated receptor γ (PPARγ)-retinoid X receptor α (RXRα) heterodimer. This structure provided the first indication of direct interactions between the DNA-binding domain (DBD) and ligand-binding domain (LBD). In this study, we investigated whether there is a similar interface between the DNA- and ligand-binding domains for the androgen receptor (AR). Despite the structural differences between the AR- and PPARγ-LBD, a combination of in silico modeling and docking pointed out a putative interface between AR-DBD and AR-LBD. The surfaces were subjected to a point mutation analysis, which was inspired by known AR mutations described in androgen insensitivity syndromes and prostate cancer. Surprisingly, AR-LBD mutations D695N, R710A, F754S, and P766A induced a decrease in DNA binding but left ligand binding unaffected, while the DBD-residing mutations K590A, K592A, and E621A lowered the ligand-binding but not the DNA-binding affinity. We therefore propose that these residues are involved in allosteric communications between the AR-DBD and AR-LBD.     

Altered transverse tubule dihydropyridine receptor binding in malignant hyperthermia

Transverse tubule (TT) membrane vesicles have been isolated from the skeletal muscle of normal and malignant hyperthermia-susceptible (MHS) pigs. MHS and normal TT did not differ in the distribution of the major proteins, cholesterol, or phospholipid content, (Na+ + K+)-ATPase activity, [3H]ouabain binding, Ca2+-ATPase activity, Mg2+-ATPase activity, or [3H]saxitoxin binding. Furthermore, in the presence of micromolar Ca2+, MHS and normal TT did not differ significantly in the KD values for either [3H]nitrendipine binding (2.7 +/- 0.6 and 3.3 +/- 0.5 nM, respectively) or (-)-[3H]desmethoxyverapamil ([3H]D888) binding (7.2 +/- 0.9 and 6.4 +/- 0.6 nM, respectively). However, in contrast to normal TT, MHS TT exhibited a significantly decreased Bmax for both [3H]nitrendipine binding (26.4 +/- 5.4 for MHS versus 40.6 +/- 3.7 pmol/mg protein for normal TT) and [3H]D888 binding (17.8 +/- 7.0 for MHS versus 37.4 +/- 5.9 pmol/mg protein for normal TT). At calcium concentrations greater than 0.1 mM, there was a greater inhibition of [3H]nitrendipine binding to normal than to MHS TT such that binding was now similar for both preparations. As with purified TT, [3H]nitrendipine binding to MHS muscle homogenates was significantly less than to normal muscle homogenates (109 +/- 20 versus 211 +/- 19 fmol/mg protein, for MHS and normal TT, respectively); this difference was not apparent when 100 mM CaCl2 was included in the binding medium. We conclude that the altered MHS TT dihydropyridine receptor properties may reflect an adaptation of the TT voltage sensing mechanism to the abnormal sarcoplasmic reticulum calcium release channel regulation in MHS muscle.     

Amyloids of Alpha-Synuclein Affect the Structure and Dynamics of Supported Lipid Bilayers

Interactions of monomeric alpha-synuclein (αS) with lipid membranes have been suggested to play an important role in initiating aggregation of αS. We have systematically analyzed the distribution and self-assembly of monomeric αS on supported lipid bilayers. We observe that at protein/lipid ratios higher than 1:10, αS forms micrometer-sized clusters, leading to observable membrane defects and decrease in lateral diffusion of both lipids and proteins. An αS deletion mutant lacking amino-acid residues 71–82 binds to membranes, but does not observably affect membrane integrity. Although this deletion mutant cannot form amyloid, significant amyloid formation is observed in the wild-type αS clusters. These results suggest that the process of amyloid formation, rather than binding of αS on membranes, is crucial in compromising membrane integrity.     

Gene expression in cortex and hippocampus during acute pneumococcal meningitis

Background  

Pneumococcal meningitis is associated with high mortality (~30%) and morbidity. Up to 50% of survivors are affected by neurological sequelae due to a wide spectrum of brain injury mainly affecting the cortex and hippocampus. Despite this significant disease burden, the genetic program that regulates the host response leading to brain damage as a consequence of bacterial meningitis is largely unknown.     

Induction of Strain-Transcending Antibodies Against Group A PfEMP1 Surface Antigens from Virulent Malaria Parasites

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.     

Role of stationary phase and eluent composition on the determination of log P values of N-hydroxyethylamide of aryloxyalkylen and pyridine carboxylic acids by reversed-phase high-performance liquid chromatography

The partition coefficients, P, between n-octanol and water of a number of growth stimulating substances, N-hydroxyethylamide of aryloxyalkylen- and pyridine carboxylic acids were obtained from Pomona College (C log P), and Rekker's (log P_Rekker) revised fragmental constant system was used to calculate log P data sets. Both of these data sets were correlated with two different substance lipophilicity parameters, log k_w and ₀. Log k_w was obtained by extrapolation of log retention factor (k) to 0% organic modifier measured in reversed-phase liquid chromatography (RPLC) systems. ₀ values were obtained from the slopes and intercepts of these relationships. The RPLC experiments were performed on four commercially available reversed-phase columns. Binary mixtures of methanol–water, methanol–phosphate buffer (pH 7.0), methanol–tricine buffer (pH 7.0) and acetonitrile–water were used as mobile phases for the determination of log k_w values. For the methanolic eluents linear regression provided satisfactory correlations (r>0.99) for the relationships log k vs. organic modifier content in the eluent, while for the acetonitrile-containing eluents a second-degree polynominal regression was necessary. For all four RPLC columns, by linear regression satisfactory correlations (r>0.99) were obtained between log k_w and log P data using methanolic eluents. In such eluents ₀ values were shown to be the second-best lipophilicity parameters. For acetonitrile-containing eluents the use of second-degree polynominal regression was necessary and, in contrast to methanol, significant influence of the applied column on regression results was observed. For acetonitrile-containing eluents the ₀-index does not provide satisfactory results for our substances. No difference in regression results between the use of buffered and non-buffered eluents was observed.     

Squalene synthase, a determinant of Raft-associated cholesterol and modulator of cancer cell proliferation

Several cues for cell proliferation, migration, and survival are transmitted through lipid rafts, membrane microdomains enriched in sphingolipids and cholesterol. Cells obtain cholesterol from the circulation but can also synthesize cholesterol de novo through the mevalonate/isoprenoid pathway. This pathway, however, has several branches and also produces non-sterol isoprenoids. Squalene synthase (SQS) is the enzyme that determines the switch toward sterol biosynthesis. Here we demonstrate that in prostate cancer cells SQS expression is enhanced by androgens, channeling intermediates of the mevalonate/isoprenoid pathway toward cholesterol synthesis. Interestingly, the resulting increase in de novo synthesis of cholesterol mainly affects the cholesterol content of lipid rafts, while leaving non-raft cholesterol levels unaffected. Conversely, RNA interference-mediated SQS inhibition results in a decrease of raft-associated cholesterol. These data show that SQS activity and de novo cholesterol synthesis are determinants of membrane microdomain-associated cholesterol in cancer cells. Remarkably, SQS knock down also attenuates proliferation and induces death of prostate cancer cells. Similar effects are observed when cancer cells are treated with the chemical SQS inhibitor zaragozic acid A. Importantly, although the anti-tumor effect of statins has previously been attributed to inhibition of protein isoprenylation, the present study shows that specific inhibition of the cholesterol biosynthesis branch of the mevalonate/isoprenoid pathway also induces cancer cell death. These findings significantly underscore the importance of de novo cholesterol synthesis for cancer cell biology and suggest that SQS is a potential novel target for antineoplastic intervention.     

Growth partitioning within beech trees (<Emphasis Type="Italic">Fagus sylvatica</Emphasis> L.) varies in response to summer heat waves and related droughts

Key message

Beech growth variability and climate sensitivity are much higher in the crown top than in the bole. The most notable bole–crown discrepancies occurred in response to extreme climate conditions.

Abstract

To characterize growth partitioning within the tree and its responses to climate, we studied eight dominant beech trees (Fagus sylvatica L.) of a pure, even-aged 98-year-old stand in Belgium. We sampled ten disks along the stem from breast height to treetop and examined the inter-annual patterns of, and discrepancies between, ring-area and volume increments by performing detailed stem analysis and dendroecological investigations. Although the common inter-annual variation among all increment series was high, we observed increasing growth variability and climate sensitivity with height, leading to notable bole–crown discrepancies. Both the common inter-annual variation and bole–crown discrepancies were mainly driven by summer heat waves and related droughts of the previous year, and spring droughts of the current year. Despite these discrepancies, the radial growth at breast height can be considered a good estimate of the tree volume increment but not for the purpose of focusing on climatic effects of isolated years. Extreme climatic conditions increase the risk of inaccurate estimations. The results of the present study are discussed in relation to tree ecophysiology hypotheses.

     

Elucidating the Aggregation Number of Dopamine-Induced α-Synuclein Oligomeric Assemblies

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson’s disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15–19 monomers and 34–38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.