Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.     

Breast MRI in nonpalpable breast lesions: a randomized trial with diagnostic and therapeutic outcome – MONET – study

Background

In orthodontic treatment, anchorage control is a fundamental aspect. Usually conventional mechanism for orthodontic anchorage control can be either extraoral or intraoral that is headgear or intermaxillary elastics. Their use are combined with various side effects such as tipping of occlusal plane or undesirable movements of teeth. Especially in cases, where key-teeth are missing, conventional anchorage defined as tooth-borne anchorage will meet limitations. Therefore, the use of endosseous implants for anchorage purposes are increasingly used to achieve positional stability and maximum anchorage.

Methods/Design

The intended study is designed as a prospective, multicenter randomized controlled trial (RCT), comparing and contrasting the effect of early loading of palatal implant therapy versus implant loading after 12 weeks post implantation using the new ortho-implant type II anchor system device (Orthosystem Straumann, Basel, Switzerland). 124 participants, mainly adult males or females, whose diagnoses require temporary stationary implant-based anchorage treatment will be randomized 1:1 to one of two treatment groups: group 1 will receive a loading of implant standard therapy after a healing period of 12 week (gold standard), whereas group 2 will receive an early loading of orthodontic implants within 1 week after implant insertion. Participants will be at least followed for 12 months after implant placement. The primary endpoint is to investigate the behavior of early loaded palatal implants in order to find out if shorter healing periods might be justified to accelerate active orthodontic treatment. Secondary outcomes will focus e.g. on achievement of orthodontic treatment goals and quantity of direct implant-bone interface of removed bone specimens. As tertiary objective, a histologic and microtomography evaluation of all retrieved implants will be performed to obtain data on the performance of the SLA surface in human bone evaluation of all retrieved implants. Additionally, resonance frequency analysis (RFA, Osstell? mentor) will be used at different times for clinically monitoring the implant stability and for histological comparison in order to measure the reliability of the resonance frequency measuring device.

Trial registration

Current Controlled Trials ISRCTN97142521.     

Formation of transient oxygen complexes of cytochrome p450 BM3 and nitric oxide synthase under high pressure

The kinetics of formation and transformation of oxygen complexes of two heme-thiolate proteins (the F393H mutant of cytochrome P450 BM3 and the oxygenase domain of endothelial nitric oxide synthase, eNOS) were studied under high pressure. For BM3, oxygen-binding characteristics (rate and activation volume) matched those measured for CO-binding. In contrast, pressure revealed a different CO- and oxygen-binding mechanism for eNOS, suggesting that it is hazardous to take CO-binding as a model for oxygen-binding. With eNOS, a ferric NO complex is formed as an intermediate in the second reaction cycle. Here we report the pressure stability of this compound. Furthermore, in the presence of 4-amino-tetrahydrobiopterin (ABH(4)), an analog to the natural second electron donor tetrahydrobiopterin (BH(4)), biphasic pressure profiles of the oxygen-binding rates were observed, both in the first and the second reaction cycles, indicative of the formation of an additional reaction intermediate. This was confirmed by experiments where ABH(4) was replaced by ABH(2), a cofactor which cannot deliver an electron. Altogether, high pressure appears to be a useful tool to characterize elementary steps in the reaction cycle of heme-thiolate proteins.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils

Degradation of agricultural land and the resulting loss of soil biodiversity and productivity are of great concern. Land-use management practices can be used to ameliorate such degradation. The soil bacterial communities at three separate arable farms in eastern England, with different farm management practices, were investigated by using a polyphasic approach combining traditional soil analyses, physiological analysis, and nucleic acid profiling. Organic farming did not necessarily result in elevated organic matter levels; instead, a strong association with increased nitrate availability was apparent. Ordination of the physiological (BIOLOG) data separated the soil bacterial communities into two clusters, determined by soil type. Denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism analyses of 16S ribosomal DNA identified three bacterial communities largely on the basis of soil type but with discrimination for pea cropping. Five fields from geographically distinct soils, with different cropping regimens, produced highly similar profiles. The active communities (16S rRNA) were further discriminated by farm location and, to some degree, by land-use practices. The results of this investigation indicated that soil type was the key factor determining bacterial community composition in these arable soils. Leguminous crops on particular soil types had a positive effect upon organic matter levels and resulted in small changes in the active bacterial population. The active population was therefore more indicative of short-term management changes.     

Population differences in spatial learning in three-spined sticklebacks

In a changing environment, learning and memory are essential for an animal''s survival and reproduction. The role played by the environment in shaping learning and memory is now attracting considerable attention. Until now, studies have tended to compare the behaviour of two, or at best a few species but interspecific comparisons can be misleading as many life history variables other than environment may differ between species. Here we report on an experiment designed to determine how learning varies between different populations of the same species, the three-spined stickleback. We found differences between the populations in their ability to solve a spatial task and also in the spatial strategies they used. A second simple learning task showed that these differences were not the result of gross differences in learning ability or adaptation to laboratory conditions. We discuss these results and suggest that the behavioural differences may relate to features of the respective habitats from which the fish were sampled.     

Reliability of an injury scoring system for horses

Background

The risk of injuries is of major concern when keeping horses in groups and there is a need for a system to record external injuries in a standardised and simple way. The objective of this study, therefore, was to develop and validate a system for injury recording in horses and to test its reliability and feasibility under field conditions.

Methods

Injuries were classified into five categories according to severity. The scoring system was tested for intra- and inter-observer agreement as well as agreement with a 'golden standard' (diagnosis established by a veterinarian). The scoring was done by 43 agricultural students who classified 40 photographs presented to them twice in a random order, 10 days apart. Attribute agreement analysis was performed using Kendall's coefficient of concordance (Kendall's W), Kendall's correlation coefficient (Kendall's τ) and Fleiss' kappa. The system was also tested on a sample of 100 horses kept in groups where injury location was recorded as well.

Results

Intra-observer agreement showed Kendall's W ranging from 0.94 to 0.99 and 86% of observers had kappa values above 0.66 (substantial agreement). Inter-observer agreement had an overall Kendall's W of 0.91 and the mean kappa value was 0.59 (moderate). Agreement for all observers versus the 'golden standard' had Kendall's τ of 0.88 and the mean kappa value was 0.66 (substantial). The system was easy to use for trained persons under field conditions. Injuries of the more serious categories were not found in the field trial.

Conclusion

The proposed injury scoring system is easy to learn and use also for people without a veterinary education, it shows high reliability, and it is clinically useful. The injury scoring system could be a valuable tool in future clinical and epidemiological studies.

     

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

Background  

Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.