Accessibility and potency of uterotonic drugs purchased by simulated clients in four districts in India.

BACKGROUND: Surveillance of drug quality for antibiotics, antiretrovirals, antimalarials and vaccines is better established than surveillance for maternal health drugs in low-income countries, particularly uterotonic drugs for the prevention and treatment of postpartum hemorrhage. The objectives of this study are to: assess private sector accessibility of four drugs used for uterotonic purposes (oxytocin, methylergometrine, misoprostol, valethamate bromide); and to assess potency of oxytocin and methylergometrine ampoules purchased by simulated clients. METHODS: The study was conducted in Hassan and Bagalkot districts in Karnataka state and Agra and Gorakhpur districts in Uttar Pradesh state. A sample of 877 private pharmacies was selected (using a stratified, systematic sampling with random start), among which 847 were successfully visited. The target sample size for assessment of accessibility was 50 pharmacies per drug, per district. The target sample size for potency assessment was 100 purchases each of oxytocin and methylergometrine across all districts. Successful drug purchases varied by state. RESULTS: In Agra and Gorakhpur, 90%-100% of visits for each of the drugs resulted in a purchase. In Bagalkot and Hassan, only 29%-52% of visits for each drug resulted in a purchase. Regarding potency, the percent of active pharmaceutical ingredient was assessed using United States Pharmacopeia monograph #33 for both drugs; 193 and 188 ampoules of oxytocin and methylergometrine, respectively, were assessed. The percent of oxytocin ampoules outside manufacturer specification ranged from 33%-40% in Karnataka and from 22%-50% in Uttar Pradesh. In Bagalkot and Hassan, 96% and 100% of the methylergometrine ampoules were outside manufacturer specification, respectively. In Agra and Gorakhpur, 54% and 44% were outside manufacturer specification, respectively. CONCLUSION: Private sector accessibility of uterotonic drugs in study districts in Karnataka warrants attention. Most importantly, interventions to assure quality oxytocin and particularly methylergometrine are needed in study districts in both states.

Engineering amyloid-like assemblies from unstructured peptides via site-specific lipid conjugation.

Aggregation of amyloid beta (Aß) into oligomers and fibrils is believed to play an important role in the development of Alzheimer's disease (AD). To gain further insight into the principles of aggregation, we have investigated the induction of ß-sheet secondary conformation from disordered native peptide sequences through lipidation, in 1-2% hexafluoroisopropanol (HFIP) in phosphate buffered saline (PBS). Several parameters, such as type and number of lipid chains, peptide sequence, peptide length and net charge, were explored keeping the ratio peptide/HFIP constant. The resulting lipoconjugates were characterized by several physico-chemical techniques: Circular Dichroism (CD), Attenuated Total Reflection InfraRed (ATR-IR), Thioflavin T (ThT) fluorescence, Dynamic Light Scattering (DLS), solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy and Electron Microscopy (EM). Our data demonstrate the generation of ß-sheet aggregates from numerous unstructured peptides under physiological pH, independent of the amino acid sequence. The amphiphilicity pattern and hydrophobicity of the scaffold were found to be key factors for their assembly into amyloid-like structures.

Importance of lipid-pore loop interface for potassium channel structure and function.

Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.

Structural determinants of specific lipid binding to potassium channels.

We have investigated specific lipid binding to the pore domain of potassium channels KcsA and chimeric KcsA-Kv1.3 on the structural and functional level using extensive coarse-grained and atomistic molecular dynamics simulations, solid-state NMR, and single channel measurements. We show that, while KcsA activity is critically modulated by the specific and cooperative binding of anionic nonannular lipids close to the channel's selectivity filter, the influence of nonannular lipid binding on KcsA-Kv1.3 is much reduced. The diminished impact of specific lipid binding on KcsA-Kv1.3 results from a point-mutation at the corresponding nonannular lipid binding site leading to a salt-bridge between adjacent KcsA-Kv1.3 subunits, which is conserved in many voltage-gated potassium channels and prevents strong nonannular lipid binding to the pore domain. Our findings elucidate how protein-lipid and protein-protein interactions modulate K(+) channel activity. The combination of MD, NMR, and functional studies as shown here may help to dissect the structural and dynamical processes that are critical for the functioning of larger membrane proteins, including Kv channels in a membrane setting.

Rapid prediction of multi-dimensional NMR data sets.

We present a computational environment for Fast Analysis of multidimensional NMR DAta Sets (FANDAS) that allows assembling multidimensional data sets from a variety of input parameters and facilitates comparing and modifying such "in silico" data sets during the various stages of the NMR data analysis. The input parameters can vary from (partial) NMR assignments directly obtained from experiments to values retrieved from in silico prediction programs. The resulting predicted data sets enable a rapid evaluation of sample labeling in light of spectral resolution and structural content, using standard NMR software such as Sparky. In addition, direct comparison to experimental data sets can be used to validate NMR assignments, distinguish different molecular components, refine structural models or other parameters derived from NMR data. The method is demonstrated in the context of solid-state NMR data obtained for the cyclic nucleotide binding domain of a bacterial cyclic nucleotide-gated channel and on membrane-embedded sensory rhodopsin II. FANDAS is freely available as web portal under WeNMR ( http://www.wenmr.eu/services/FANDAS ).

Supramolecular structure of membrane-associated polypeptides by combining solid-state NMR and molecular dynamics simulations.

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone. Our study focuses on the Shaker B ball peptide that is representative for rapid N-type inactivating domains of voltage-gated K(+) channels, associated with negatively charged lipid bilayers.

Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel.

Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.

Fractional deuteration applied to biomolecular solid-state NMR spectroscopy.

Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and D(2)O medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at H(α) and H(ß) positions reduce spectral congestion in ((1)H,(13)C,(15)N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR.

Structural characterization of polyglutamine fibrils by solid-state NMR spectroscopy.

Protein aggregation via polyglutamine stretches occurs in a number of severe neurodegenerative diseases such as Huntington's disease. We have investigated fibrillar aggregates of polyglutamine peptides below, at, and above the toxicity limit of around 37 glutamine residues using solid-state NMR and electron microscopy. Experimental data are consistent with a dry fibril core of at least 70-80 Å in width for all constructs. Solid-state NMR dipolar correlation experiments reveal a largely ß-strand character of all samples and point to tight interdigitation of hydrogen-bonded glutamine side chains from different sheets. Two approximately equally frequent populations of glutamine residues with distinct sets of chemical shifts are found, consistent with local backbone dihedral angles compensating for ß-strand twist or with two distinct sets of side-chain conformations. Peptides comprising 15 glutamine residues are present as single extended ß-strands. Data obtained for longer constructs are most compatible with a superpleated arrangement with individual molecules contributing ß-strands to more than one sheet and an antiparallel assembly of strands within ß-sheets.

Sequence-independent control of peptide conformation in liposomal vaccines for targeting protein misfolding diseases.

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.