Observation and kinematic description of long actin tracks induced by spherical beads

We report an in vitro study comparing the growth of long actin tails induced by spherical beads coated with the verprolin central acidic domain of the polymerization enzyme N-WASP to that induced by Listeria monocytogenes in similar cellular extracts. The tracks behind the beads show characteristic differences in shape and curvature from those left by the bacteria, which have an elongated shape and a similar polymerization-inducing enzyme distributed only on the rear surface of the cell. The experimental tracks are simulated using a generalized kinematic model, which incorporates three modes of bead rotation with respect to the tail. The results show that the trajectories of spherical beads are mechanically deterministic rather than random, as suggested by stochastic models. Assessment of the bead rotation and its mechanistic basis offers insights into the biological function of actin-based motility.     

Crystallographic studies of the complexes of antiviral protein griffithsin with glucose and N-acetylglucosamine

Crystal structures of complexes of an antiviral lectin griffithsin (GRFT) with glucose and N-acetylglucosamine were solved and refined at high resolution. In both complexes, all six monosaccharide-binding sites of GRFT were occupied and the mode of binding was similar to that of mannose. In our previous attempts to obtain a complex with N-acetylglucosamine by soaking, only a single site was occupied; thus, cocrystallization was clearly superior despite lower concentration of the ligand. Isothermal titration calorimetric experiments with N-acetylglucosamine, glucose, and mannose provided enthalpic evidence of distinct binding differences between the three monosaccharides. A comparison of the mode of binding of different monosaccharides is discussed in the context of the antiviral activity of GRFT, based on specific binding to high-mannose-containing complex carbohydrates found on viral envelopes.     

A long-term "memory" of HIF induction in response to chronic mild decreased oxygen after oxygen normalization

Background

Cardiovascular diseases (CVD) are the leading cause of death and the third cause of disability in Europe. Prevention programmes should include interventions aimed at a reduction of medical risk factors (hypertension, hypercholesterol, hyperglycemia, overweight and obesity) as well as behavioural risk factors (sedentary lifestyle, high fat intake and low fruit and vegetable intake, smoking). The aim of this study is to investigate the effects of a multifaceted, multidisciplinary electronic prevention programme on cardiovascular risk factors.

Methods/Design

In a randomized controlled trial, one group will receive a maximal intervention (= intervention group). The intervention group will be compared to the control group receiving a minimal intervention. An inclusion of 350 patients in total, with a follow-up of 3 years is foreseen. The inclusion criteria are age between 25–65 and insured by the Onderlinge Ziekenkas, insuring for guaranteed income in case of illness for self-employed. The maximal intervention group receives several prevention consultations by their general practitioner (GP) using a new type of cardiovascular risk calculator with personalised feedback on behavioural risk factors. These patients receive a follow-up with intensive support of health behaviour change via different methods, i.e. a tailored website and personal advice of a multidisciplinary team (psychologist, physiotherapist and dietician). The aim of this strategy is to reduce cardiovascular risk factors according to the guidelines. The primary outcome measures will be cardiovascular risk factors. The secondary outcome measures are cardiovascular events, quality of life, costs and incremental cost effectiveness ratios. The control group receives prevention consultations using a new type of cardiovascular risk calculator and general feedback.

Discussion

This trial incorporates interventions by GPs and other health professionals aiming at a reduction of medical and behavioural cardiovascular risk factors. An assessment of clinical, psychological and economical outcome measures will be performed.

Trial registration

ISRCTN23940498     

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.     

Early dysregulation of the mitochondrial proteome in a mouse model of Alzheimer's disease

Mitochondrial structural and functional alterations appear to play to an important role in the pathogenesis of Alzheimer's disease (AD). In the present study, we used a quantitative comparative proteomic profiling approach to analyze changes in the mitochondrial proteome in AD. A triple transgenic mouse model of AD (3xTg-AD) which harbors mutations in three human transgenes, APP(Swe), PS1(M146V) and Tau(P301L), was used in these experiments. Quantitative differences in the mitochondrial proteome between the cerebral cortices of 6-month-old male 3xTg-AD and non-transgenic mice were determined by using two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass spectrometry. We identified 23 different proteins whose expression levels differed significantly between triple transgenic and non-transgenic mitochondria. Both down-regulated and up-regulated mitochondrial proteins were observed in transgenic AD cortices. Proteins which were dysregulated in 3xTg-AD cortices functioned in a wide variety of metabolic pathways, including the citric acid cycle, oxidative phosphorylation, pyruvate metabolism, glycolysis, oxidative stress, fatty acid oxidation, ketone body metabolism, ion transport, apoptosis, and mitochondrial protein synthesis. These alterations in the mitochondrial proteome of the cerebral cortices of triple transgenic AD mice occurred before the development of significant amyloid plaque and neurofibrillary tangles, indicating that mitochondrial dysregulation is an early event in AD.     

Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.     

An adenylyl cyclase pseudogene in Mycobacterium tuberculosis has a functional ortholog in Mycobacterium avium

A number of genes similar to mammalian Class III nucleotide cyclases are found in mycobacteria, and biochemical characterization of some of these proteins has indicated that they code for adenylyl cyclases, with properties similar to the mammalian enzymes. Our earlier bioinformatic analysis had predicted that the Rv1120c gene in Mycobacterium tuberculosis is a pseudogene, while analysis of the genome of Mycobacterium avium indicated the presence of a functional ortholog. We therefore cloned and expressed Rv1120c and its ortholog from M. avium, Ma1120, in Escherichia coli, and find that while the protein from M. tuberculosis is misfolded and found in inclusion bodies, Ma1120 is expressed to high levels as a functional adenylyl cyclase. Sequence analysis of Ma1120 indicates interesting variations in critical amino acids that are known to be important for catalytic activity. Ma1120 is maximally active in the presence of MnATP as substrate ((app)Km approximately 400 microM), and is inhibited by P-site inhibitors (IC50 of 2',5'-dideoxy-3'-adenosine triphosphate approximately 730 nM) and tyrphostins (IC50 approximately 36 microM) in a manner similar to the mammalian enzymes. This therefore represents the first Class III cyclase biochemically characterized from M. avium, and the absence of a functional ortholog in M. tuberculosis suggests a unique role for this enzyme in M. avium.     

Hypocitraturia despite potassium citrate tablet supplementation

Citrate supplementation is widely used in the prevention of recurrent nephrolithiasis with hypocitraturia. Potassium citrate is the most commonly used citrate agent for this indication. In patients with chronic diarrheal syndromes, the absorption of potassium citrate can be affected. We describe a patient who presented with recurrent nephrolithiasis and chronic diarrhea and was found to have severe hypocitraturia despite citrate supplementation with potassium citrate tablets, likely due to inadequate gastrointestinal absorption of citrate from the slow-release wax-matrix tablets.     

Synthesis and biological activity of N4-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic and antitumor agents

A series of eight N⁴-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8–15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N⁴-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16–20 were synthesized to evaluate the importance of the 2-NH₂ moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of α-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of α-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40–42, respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8–20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.