Inversion of the anomeric configuration of the transferred sugar during inactivation of the macrolide antibiotic oleandomycin catalyzed by a macrolide glycosyltransferase

AbstractMacrolides are a group of antibiotics structurally characterized by a macrocyclic lactone to which one or several deoxy-sugar moieties are attached. The sugar moieties are transferred to the different aglycones by glycosyltransferases (GTF). The OleI GTF of an oleandomycin producer, Streptomyces antibioticus, catalyzes the inactivation of this macrolide by glycosylation. The product of this reaction was isolated and its structure elucidated. The donor substrate of the reaction was UDP-α-D-glucose, but the reaction product showed a β-glycosidic linkage. The inversion of the anomeric configuration of the transferred sugar and other data about the kinetics of the reaction and primary structure analysis of several GTFs are compatible with a reaction mechanism involving a single nucleophilic substitution at the sugar anomeric carbon in the catalytic center of the enzyme

Pulse Sequences for NMR Quantum Computers: How to Manipulate Nuclear Spins While Freezing the Motion of Coupled Neighbours

We show how to divide a coupled multi-spin system into a small subset of ``active'' spins that evolve under chemical shift or scalar coupling operators, and a larger subset of ``spectator'' spins which are returned to their initial states, as if their motion had been temporarily frozen. This allows us to implement basic one-qubit and two-qubit operations from which general operations on $N$-qubits can be constructed, suitable for quantum computation. The principles are illustrated by experiments on the three coupled protons of 2,3-dibromopropanoic acid, but the method is applicable to any spin-1/2 nuclei and to systems containing arbitrary numbers of coupled spins.Comment: 11 pages, 5 fig

In Vivo Detection of Perinatal Brain Metabolite Changes in a Rabbit Model of Intrauterine Growth Restriction (IUGR)

Background Intrauterine growth restriction (IUGR) is a risk factor for abnormal neurodevelopment.We studied a rabbit model of IUGR by magnetic resonance imaging (MRI) and spectroscopy (MRS), to assess in vivo brain structural and metabolic consequences, and identify potential metabolic biomarkers for clinical translation. Methods IUGR was induced in 3 pregnant rabbits at gestational day 25, by 40-50% uteroplacental vessel ligation in one horn; the contralateral horn was used as control. Fetuses were delivered at day 30 and weighted. A total of 6 controls and 5 IUGR pups underwent T2-w MRI and localized proton MRS within the first 8 hours of life, at 7T. Changes in brain tissue volumes and respective contributions to each MRS voxel were estimated by semi-automated registration of MRI images with a digital atlas of the rabbit brain. MRS data were used for: (i) absolute metabolite quantifications, using linear fitting; (ii) local temperature estimations, based on the water chemical shift; and (iii) classification, using spectral pattern analysis. Results Lower birth weight was associated with (i) smaller brain sizes, (ii) slightly lower brain temperatures, and (iii) differential metabolite profile changes in specific regions of the brain parenchyma. Specifically, we found estimated lower levels of aspartate and N-acetylaspartate (NAA) in the cerebral cortex and hippocampus (suggesting neuronal impairment), and higher glycine levels in the striatum (possible marker of brain injury). Our results also suggest that the metabolic changes in cortical regions are more prevalent than those detected in hippocampus and striatum. Conclusions IUGR was associated with brain metabolic changes in vivo, which correlate well with the neurostructural changes and neurodevelopment problems described in IUGR. Metabolic parameters could constitute non invasive biomarkers for the diagnosis and abnormal neurodevelopment of perinatal origin

A Kernel for Open Source Drug Discovery in Tropical Diseases

Open source drug discovery, a promising alternative avenue to conventional patent-based drug development, has so far remained elusive with few exceptions. A major stumbling block has been the absence of a critical mass of preexisting work that volunteers can improve through a series of granular contributions. This paper introduces the results from a newly assembled computational pipeline for identifying protein targets for drug discovery in ten organisms that cause tropical diseases. We have also experimentally tested two promising targets for their binding to commercially available drugs, validating one and invalidating the other. The resulting kernel provides a base of drug targets and lead candidates around which an open source community can nucleate. We invite readers to donate their judgment and in silico and in vitro experiments to develop these targets to the point where drug optimization can begin

NMR for chemists and biologists

This book offers a concise introduction to the field of nuclear magnetic resonance or NMR. It presents the basic foundations of NMR in a non-mathematical way and provides an overview of both recent and important biological applications of NMR

Characterization of dequalinium as a XIAP antagonist that targets the BIR2 domain

8 páginas, 5 figuras, 1 tabla. PMID:21340509[PubMed]Inhibitor of apoptosis proteins (IAPs) regulate the activity of caspases in apoptosis. The human X chromosome-encoded IAP (XIAP) is one of the more potent members of the IAP family and it has been described as a central regulator of apoptosis. Thus, molecules that inhibit XIAP could offer therapeutic opportunities to treat unwanted apoptosis inhibition. In the present study we have applied the selective optimization of side activities (SOSA) approach to the discovery of XIAP inhibitors. In this sense, we have identified dequalinium hydrochloride (Dq) as an inhibitor of the XIAP/caspase-3 interaction both in vitro and in cellular assaysThis work was supported by grants from the Spanish Ministry of Science and Innovation (MICINN - BIO2007-60066), Generalitat Valenciana Prometeo 2010/005 (partially funded with ERDF) and Consolider-Ingenio 2010 (MICINN - CSD2008-00005C) to E.P.-P. We thank Susana Rubio and Alicia García-Jareño for technical assistance. Y. P.-R. was supported by a postdoctoral fellowship from National Autonomous University of Mexico and Consejo Superior de Investigaciones Científicas of Spain (UNAM-CSIC).Peer reviewe

Metabolomic Profile of Umbilical Cord Blood Plasma from Early and Late Intrauterine Growth Restricted (IUGR) Neonates with and without Signs of Brain Vasodilation

Objectives: To characterize via NMR spectroscopy the full spectrum of metabolic changes in umbilical vein blood plasma of newborns diagnosed with different clinical forms of intrauterine growth restriction (IUGR). Methods: 23 early IUGR cases and matched 23 adequate-for-gestational-age (AGA) controls and 56 late IUGR cases with 56 matched AGAs were included in this study. Early IUGR was defined as a birth weight 35 weeks. This group was subdivided in 18 vasodilated (VD) and 38 non-VD late IUGR fetuses. All AGA patients had a birth weight >10th centile. 1H nuclear magnetic resonance (NMR) metabolomics of the blood samples collected from the umbilical vein at delivery was obtained. Multivariate statistical analysis identified several metabolites that allowed the discrimination between the different IUGR subgroups, and their comparative levels were quantified from the NMR data. Results: The NMR-based analysis showed increased unsaturated lipids and VLDL levels in both early and late IUGR samples, decreased glucose and increased acetone levels in early IUGR. Non-significant trends for decreased glucose and increased acetone levels were present in late IUGR, which followed a severity gradient when the VD and non-VD subgroups were considered. Regarding amino acids and derivatives, early IUGR showed significantly increased glutamine and creatine levels, whereas the amounts of phenylalanine and tyrosine were decreased in early and late-VD IUGR samples. Valine and leucine were decreased in late IUGR samples. Choline levels were decreased in all clinical subforms of IUGR. Conclusions: IUGR is not associated with a unique metabolic profile, but important changes are present in different clinical subsets used in research and clinical practice. These results may help in characterizing comprehensively specific alterations underlying different IUGR subsets

Production of heparanase constructs suitable for nuclear magnetic resonance and drug discovery studies

Heparanase is an endo-β-D-glucosidase capable of specifically degrading heparan sulphate, one of the main components of the extracellular matrix. This 65 kDa polypeptide is implicated in cancer processes such as tumour formation, angiogenesis and metastasis, making it a very attractive target in antitumour treatments. Structure-based approaches to find inhibitors of heparanase have been historically hampered by the lack of success in crystallizing the protein. With the aim to undertake the NMR structural characterisation of heparanase, we have designed and produced, using recombinant methods, smaller constructs of heparanase containing the catalytically active glutamic acids and the two binding sites for heparan sulphate. An extensive range of expression and purification conditions were evaluated to alleviate the intrinsic low solubility and aggregation propensity of heparanase, allowing the obtention of the enzyme in milligram quantities, both unlabelled and ¹⁵N-labelled for NMR studies. Using the smallest of the designed constructs and applying NMR and SPR methodologies, we have demonstrated that known inhibitors of heparanase bind to this construct specifically and selectively with K(D) values in the range of those reported for human heparanase, validating it for future drug discovery projects focused on the identification of novel inhibitors of this enzyme