Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

Systematic Review of the Measurement Properties of Tools Used to Measure Behaviour Problems in Young Children with Autism

Background

Behaviour problems are common in young children with autism spectrum disorder (ASD). There are many different tools used to measure behavior problems but little is known about their validity for the population.

Objectives

To evaluate the measurement properties of behaviour problems tools used in evaluation of intervention or observational research studies with children with ASD up to the age of six years.

Methods

Behaviour measurement tools were identified as part of a larger, two stage, systematic review. First, sixteen major electronic databases, as well as grey literature and research registers were searched, and tools used listed and categorized. Second, using methodological filters, we searched for articles examining the measurement properties of the tools in use with young children with ASD in ERIC, MEDLINE, EMBASE, CINAHL, and PsycINFO. The quality of these papers was then evaluated using the COSMIN checklist.

Results

We identified twelve tools which had been used to measure behaviour problems in young children with ASD, and fifteen studies which investigated the measurement properties of six of these tools. There was no evidence available for the remaining six tools. Two questionnaires were found to be the most robust in their measurement properties, the Child Behavior Checklist and the Home Situations Questionnaire—Pervasive Developmental Disorders version.

Conclusions

We found patchy evidence on reliability and validity, for only a few of the tools used to measure behaviour problems in young children with ASD. More systematic research is required on measurement properties of tools for use in this population, in particular to establish responsiveness to change which is essential in measurement of outcomes of intervention.

PROSPERO Registration Number

CRD42012002223     

Phenomenological partial-specific volumes for G-quadruplex DNAs

Accurate partial-specific volume ([`(v)] \overline{v} ) values are required for sedimentation velocity and sedimentation equilibrium analyses. For nucleic acids, the estimation of these values is complicated by the fact that [`(v)] \overline{v} depends on base composition, secondary structure, solvation and the concentrations and identities of ions in the surrounding buffer. Here we describe sedimentation equilibrium measurements of the apparent isopotential partial-specific volume ϕ′ for two G-quadruplex DNAs and a single-stranded DNA of similar molecular weight and base composition. The G-quadruplex DNAs are a 22 nucleotide fragment of the human telomere consensus sequence and a 27 nucleotide fragment from the human c-myc promoter. The single-stranded DNA is 26 nucleotides long and is designed to have low propensity to form secondary structures. Parallel measurements were made in buffers containing NaCl and in buffers containing KCl, spanning the range 0.09 M ≤ [salt] ≤ 2.3 M. Limiting values of ϕ′, extrapolated to [salt] = 0 M, were: 22-mer (NaCl-form), 0.525 ± 0.004 mL/g; 22-mer (KCl-form), 0.531 ± 0.006 mL/g; 27-mer (NaCl-form), 0.548 ± 0.005 mL/g; 27-mer (KCl-form), 0.557 ± 0.006 mL/g; 26-mer (NaCl-form), 0.555 ± 0.004 mL/g; 26-mer (KCl-form), 0.564 ± 0.006 mL/g. Small changes in ϕ′ with [salt] suggest that large changes in counterion association or hydration are unlikely to take place over these concentration ranges.     

Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase

Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the basis of crystal structures and previous mapping studies, four sites (Glu-469/Arg-470, Met-490/Arg-491, His-495/Asn-496, and Arg-498/Thr-499; thimet oligopeptidase residues listed first) in their substrate-binding channels appear positioned to account for differences in specificity. Thimet oligopeptidase mutated so that neurolysin residues are at all four positions cleaves neurotensin at the neurolysin site, and the reverse mutations in neurolysin switch hydrolysis to the thimet oligopeptidase site. Using a series of constructs mutated at just three of the sites, it was determined that mutations at only two (Glu-469/Arg-470 and Arg-498/Thr-499) are required to swap specificity, a result that was confirmed by testing the two-mutant constructs. If only either one of the two sites is mutated in thimet oligopeptidase, then the enzyme cleaves almost equally at the two hydrolysis positions. Crystal structures of both two-mutant constructs show that the mutations do not perturb local structure, but side chain conformations at the Arg-498/Thr-499 position differ from those of the mimicked enzyme. A model for differential recognition of neurotensin based on differences in surface charge distribution in the substrate binding sites is proposed. The model is supported by the finding that reducing the positive charge on the peptide results in cleavage at both hydrolysis sites.     

SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis

A progressive loss of neurons with age underlies a variety of debilitating neurological disorders, including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), yet few effective treatments are currently available. The SIR2 gene promotes longevity in a variety of organisms and may underlie the health benefits of caloric restriction, a diet that delays aging and neurodegeneration in mammals. Here, we report that a human homologue of SIR2, SIRT1, is upregulated in mouse models for AD, ALS and in primary neurons challenged with neurotoxic insults. In cell-based models for AD/tauopathies and ALS, SIRT1 and resveratrol, a SIRT1-activating molecule, both promote neuronal survival. In the inducible p25 transgenic mouse, a model of AD and tauopathies, resveratrol reduced neurodegeneration in the hippocampus, prevented learning impairment, and decreased the acetylation of the known SIRT1 substrates PGC-1alpha and p53. Furthermore, injection of SIRT1 lentivirus in the hippocampus of p25 transgenic mice conferred significant protection against neurodegeneration. Thus, SIRT1 constitutes a unique molecular link between aging and human neurodegenerative disorders and provides a promising avenue for therapeutic intervention.     

Organic carbon and nitrogen removal from a strong wastewater using a denitrifying suspended growth reactor and a horizontal-flow biofilm reactor

Recirculation of fully nitrified effluent from a laboratory horizontal-flow biofilm reactor (HFBR) to a mixed pre-denitrification reactor (DR) was used to remove organic carbon and nitrogen from synthetic dairy wastewater. Three recirculation ratios of 2, 4, and 6 were examined in this study and the average filtered chemical oxygen demand (CODf) and total nitrogen (TN) removals were up to 97.4% and 85.5%, respectively, at 11 degrees C. In the DR, the nitrate nitrogen removal efficiencies and rates were 86-96% and 22-34 g N/m3 d. In the HFBR, the ammonium nitrogen removal rates were 293-337 mg N/m2 d.     

Computational modeling of factors that modulate the unique FeNO bonding in {FeNO}6 heme–thiolate model complexes

A density functional theory account of the changes in FeNO bonding that occur in response to both bonded and nonbonded structural perturbations is reported for a series of {FeNO}(6) heme-thiolate model complexes. Using [Fe(porphine)(SCH(3))NO] as the reference complex, we constructed models to mimic equatorial (cis), distal, and proximal influences of protein environments. Overall, the results from these calculations reveal that the Fe-NO and N-O bond strengths change in the same direction upon variations in structure and environment. These bonding changes are manifested in unique direct correlations between the Fe-NO and N-O vibrational frequencies and bond lengths, as evidenced by their positive slopes (slopes of the familiar inverse or backbonding correlations are negative). The electronic origin of the direct correlations appears to derive from the electron density distribution in high-energy molecular orbitals. This variability modulates the FeNO antibonding character throughout the triatomic FeNO moiety. The results of this study suggest that the stabilities and reactivities of {FeNO}(6) centers in heme-thiolate enzymes can be modulated over a significant range through a variety of bonded and nonbonded means.     

Canopy interactions and physical stress gradients in subtidal communities

Species interactions are integral drivers of community structure and can change from competitive to facilitative with increasing environmental stress. In subtidal marine ecosystems, however, interactions along physical stress gradients have seldom been tested. We observed seaweed canopy interactions across depth and latitudinal gradients to test whether light and temperature stress structured interaction patterns. We also quantified interspecific and intraspecific interactions among nine subtidal canopy seaweed species across three continents to examine the general nature of interactions in subtidal systems under low consumer pressure. We reveal that positive and neutral interactions are widespread throughout global seaweed communities and the nature of interactions can change from competitive to facilitative with increasing light stress in shallow marine systems. These findings provide support for the stress gradient hypothesis within subtidal seaweed communities and highlight the importance of canopy interactions for the maintenance of subtidal marine habitats experiencing environmental stress.