Structural changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding studied by fourier transform infrared spectroscopy

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding were recorded in H(2)O and (2)H(2)O at -7 degrees C and pH 7.0. The reaction cycle was triggered by the photochemical release of nucleotides (ATP, ADP, and AMP-PNP) from a biologically inactive precursor (caged ATP, P(3)-1-(2-nitrophenyl) adenosine 5'-triphosphate, and related caged compounds). Infrared absorbance changes due to ATP release and two steps of the Ca(2+)-ATPase reaction cycle, ATP binding and phosphorylation, were followed in real time. Under the conditions used in our experiments, the rate of ATP binding was limited by the rate of ATP release (k(app) congruent with 3 s(-1) in H(2)O and k(app) congruent with 7 s(-1) in (2)H(2)O). Bands in the amide I and II regions of the infrared spectrum show that the conformation of the Ca(2+)-ATPase changes upon nucleotide binding. The observation of bands in the amide I region can be assigned to perturbations of alpha-helical and beta-sheet structures. According to similar band profiles in the nucleotide binding spectra, ATP, AMP-PNP, and ADP induce similar conformational changes. However, subtle differences between ATP and AMP-PNP are observed; these are most likely due to the protonation state of the gamma-phosphate group. Differences between the ATP and ADP binding spectra indicate the significance of the gamma-phosphate group in the interactions between the Ca(2+)-ATPase and the nucleotide. Nucleotide binding affects Asp or Glu residues, and bands characteristic of their protonated side chains are observed at 1716 cm(-1) (H(2)O) and 1706 cm(-1) ((2)H(2)O) and seem to depend on the charge of the phosphate groups. Bands at 1516 cm(-1) (H(2)O) and 1514 cm(-1) ((2)H(2)O) are tentatively assigned to a protonated Tyr residue affected by nucleotide binding. Possible changes in Arg, Trp, and Lys absorption and in the nucleoside are discussed. The spectra are compared with those of nucleotide binding to arginine kinase, creatine kinase, and H-ras P21.     

Substrate binding and enzyme function investigated by infrared spectroscopy

Protein conformational changes triggered by molecule binding are increasingly investigated by infrared spectroscopy often using caged compounds. Several examples of molecule-protein recognition studies are given, which focus on nucleotide binding to proteins. The investigation of enzyme mechanisms is illustrated in detail using the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane as an example. It is shown that infrared spectroscopy provides valuable information on general aspects of enzyme function as well as on molecular details of molecule-protein interactions and the mechanism of catalysis.     

Functional Characterization of the Conserved “GLK” Motif in Mitochondrial Porin from Neurospora crassa

Mitochondrial porin facilitates the diffusion of small hydrophilic molecules across the mitochondrial outer membrane. Despite low sequence similarity among porins from different species, a glycine-leucine-lysine (GLK) motif is conserved in mitochondrial and Neisseria porins. To investigate the possible roles of these conserved residues, including their hypothesized participation in ATP binding by the protein, we replaced the lysine residue of the GLK motif of Neurospora crassa porin with glutamic acid through site-directed mutagenesis of the corresponding gene. Although the pores formed by this protein have size and gating characteristics similar to those of the wild-type protein, the channels formed by GLEporin are less anion selective than the wild-type pores. The GLEporin retains the ability to be cross linked to [-³²P]ATP, indicating that the GLK sequence is not essential for ATP binding. Furthermore, the pores formed by both GLEporin and the wild-type protein become more cation selective in the presence of ATP. Taken together, these results support structural models that place the GLK motif in a part of the ion-selective -barrel that is not directly involved in ATP binding.     

Evolution of metabolic diversity: Insights from microbial polyketide synthases

Polyketides are a family of complex natural products that are built from simple carboxylic acid building blocks. In microorganisms, the majority of these secondary metabolites are produced by exceptionally large, multifunctional proteins termed polyketide synthases (PKSs). Each unit of a type I PKS assembly line resembles a mammalian type fatty acid synthase (FAS), although certain domains are optionally missing. The evolutionary analysis of microbial PKS has revealed a long joint evolution process of PKSs and FASs. The phylogenomic analysis of modular type I PKSs as the most widespread PKS type in bacteria showed a large impact of gene duplications and gene losses on the evolution of type I PKS in different bacterial groups. The majority of type I PKSs in actinobacteria and cyanobacteria may have evolved from a common ancestor, whereas in proteobacteria most type I PKSs were acquired from other bacterial groups. The modularization of type I PKSs almost unexceptionally started with multiple duplications of a single ancestor module. The repeating modules represent ideal platforms for recombination events that can lead to corresponding changes in the actual chemistry of the products. The analysis of these “natural reprogramming” events of PKSs may assist in the development of concepts for the biocombinatorial design of bioactive compounds.     

Fallback foraging as a way of life: Using dietary toughness to compare the fallback signal among capuchins and implications for interpreting morphological variation

The genus Cebus is one of the best extant models for examining the role of fallback foods in primate evolution. Cebus includes the tufted capuchins, which exhibit skeletal features for the exploitation of hard and tough foods. Paradoxically, these seemingly “specialized” taxa belong to the most ubiquitous group of closely related primates in South America, thriving in a range of different habitats. This appears to be a consequence of their ability to exploit obdurate fallback foods. Here we compare the toughness of foods exploited by two tufted capuchin species at two ecologically distinct sites; C. apella in a tropical rainforest, and C. libidinosus in a cerrado forest. We include dietary data for one untufted species (C. olivaceus) to assess the degree of difference between the tufted species. These data, along with information on skeletal morphology, are used to address whether or not a fallback foraging species exhibits a given suite of morphological and behavioral attributes, regardless of habitat. Both tufted species ingest and masticate a number of exceedingly tough plant tissues that appear to be used as fallback resources, however, C. libidinosus has the toughest diet both in terms of median and maximal values. Morphologically, C. libidinosus is intermediate in absolute symphyseal and mandibular measurements, and in measures of postcranial robusticity, but exhibits a higher intermembral index than C. apella. We propose that this incongruence between dietary toughness and skeletal morphology is the consequence of C. libidinosus' use of tools while on the ground for the exploitation of fallback foods. Am J Phys Anthropol 140:687–699, 2009. © 2009 Wiley-Liss, Inc.     

γ-H2AX as a Marker for Dose Deposition in the Brain of Wistar Rats after Synchrotron Microbeam Radiation

Objective

Synchrotron radiation has shown high therapeutic potential in small animal models of malignant brain tumours. However, more studies are needed to understand the radiobiological effects caused by the delivery of high doses of spatially fractionated x-rays in tissue. The purpose of this study was to explore the use of the γ-H2AX antibody as a marker for dose deposition in the brain of rats after synchrotron microbeam radiation therapy (MRT).

Methods

Normal and tumour-bearing Wistar rats were exposed to 35, 70 or 350 Gy of MRT to their right cerebral hemisphere. The brains were extracted either at 4 or 8 hours after irradiation and immediately placed in formalin. Sections of paraffin-embedded tissue were incubated with anti γ-H2AX primary antibody.

Results

While the presence of the C6 glioma does not seem to modulate the formation of γ-H2AX in normal tissue, the irradiation dose and the recovery versus time are the most important factors affecting the development of γ-H2AX foci. Our results also suggest that doses of 350 Gy can trigger the release of bystander signals that significantly amplify the DNA damage caused by radiation and that the γ-H2AX biomarker does not only represent DNA damage produced by radiation, but also damage caused by bystander effects.

Conclusion

In conclusion, we suggest that the γ-H2AX foci should be used as biomarker for targeted and non-targeted DNA damage after synchrotron radiation rather than a tool to measure the actual physical doses.     

Process evaluation of a tailored intervention programme of cardiovascular risk management in general practices

Background

A tailored implementation programme to improve cardiovascular risk management (CVRM) in general practice had little impact on outcomes. The questions in this process evaluation concerned (1) impact on counselling skills and CVRM knowledge of practice nurses, (2) their use of the various components of the intervention programme and adoption of recommended practices and (3) patients’ perceptions of counselling for CVRM.

Methods

A mixed-methods process evaluation was conducted. We assessed practice nurses’ motivational interviewing skills on audio-taped consultations using Motivational Interviewing Treatment Integrity (MITI). They also completed a clinical knowledge test. Both practice nurses and patients reported on their experiences in a written questionnaire and interviews. A multilevel regression analysis and an independent sample t test were used to examine motivational interviewing skills and CVRM knowledge. Framework analysis was applied to analyse qualitative data.

Results

Data from 34 general practices were available, 19 intervention practices and 14 control practices. No improvements were measured on motivational interviewing skills in both groups. There appeared to be better knowledge of CVRM in the control group. On average half of the practice nurses indicated that they adopted the recommended interventions, but stated that they did not necessarily record this in patients’ medical files. The tailored programme was perceived as too large. Time, follow-up support and reminders were felt to be lacking. About 20% of patients in the intervention group visited the general practice during the intervention period, yet only a small number of these patients were referred to recommended options.

Conclusions

The tailored programme was only partly used by practice nurses and had little impact on either their clinical knowledge and communication skills or on patient reported healthcare. If the assumed logical model of change is valid, a more intensive programme is needed to have an impact on CVRM in general practice at all.

     

Oral Migalastat HCl Leads to Greater Systemic Exposure and Tissue Levels of Active α-Galactosidase A in Fabry Patients when Co-Administered with Infused Agalsidase

Migalastat HCl (AT1001, 1-Deoxygalactonojirimycin) is an investigational pharmacological chaperone for the treatment of α-galactosidase A (α-Gal A) deficiency, which leads to Fabry disease, an X-linked, lysosomal storage disorder. The currently approved, biologics-based therapy for Fabry disease is enzyme replacement therapy (ERT) with either agalsidase alfa (Replagal) or agalsidase beta (Fabrazyme). Based on preclinical data, migalastat HCl in combination with agalsidase is expected to result in the pharmacokinetic (PK) enhancement of agalsidase in plasma by increasing the systemic exposure of active agalsidase, thereby leading to increased cellular levels in disease-relevant tissues. This Phase 2a study design consisted of an open-label, fixed-treatment sequence that evaluated the effects of single oral doses of 150 mg or 450 mg migalastat HCl on the PK and tissue levels of intravenously infused agalsidase (0.2, 0.5, or 1.0 mg/kg) in male Fabry patients. As expected, intravenous administration of agalsidase alone resulted in increased α-Gal A activity in plasma, skin, and peripheral blood mononuclear cells (PBMCs) compared to baseline. Following co-administration of migalastat HCl and agalsidase, α-Gal A activity in plasma was further significantly increased 1.2- to 5.1-fold compared to agalsidase administration alone, in 22 of 23 patients (95.6%). Importantly, similar increases in skin and PBMC α-Gal A activity were seen following co-administration of migalastat HCl and agalsidase. The effects were not related to the administered migalastat HCl dose, as the 150 mg dose of migalastat HCl increased α-Gal A activity to the same extent as the 450 mg dose. Conversely, agalsidase had no effect on the plasma PK of migalastat. No migalastat HCl-related adverse events or drug-related tolerability issues were identified.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01196871","term_id":"NCT01196871"}}NCT01196871     

GARN: Sampling RNA 3D Structure Space with Game Theory and Knowledge-Based Scoring Strategies

Cellular processes involve large numbers of RNA molecules. The functions of these RNA molecules and their binding to molecular machines are highly dependent on their 3D structures. One of the key challenges in RNA structure prediction and modeling is predicting the spatial arrangement of the various structural elements of RNA. As RNA folding is generally hierarchical, methods involving coarse-grained models hold great promise for this purpose. We present here a novel coarse-grained method for sampling, based on game theory and knowledge-based potentials. This strategy, GARN (Game Algorithm for RNa sampling), is often much faster than previously described techniques and generates large sets of solutions closely resembling the native structure. GARN is thus a suitable starting point for the molecular modeling of large RNAs, particularly those with experimental constraints. GARN is available from: http://garn.lri.fr/.