Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients.     

Thermally induced alkylation of diamond

We present an approach for the thermally activated formation of alkene-derived self-assembled monolayers on oxygen-terminated single and polycrystalline diamond surfaces. Chemical modification of the oxygen and hydrogen plasma-treated samples was achieved by heating in 1-octadecene. The resulting layers were characterized using X-ray photoelectron spectroscopy, thermal desorption spectroscopy, atomic force microscopy, Fourier transform infrared spectroscopy, and water contact angle measurements. This investigation reveals that alkenes selectively attach to the oxygen-terminated sites via covalent C-O-C bonds. The hydrophilic oxygen-terminated diamond is rendered strongly hydrophobic following this reaction. The nature of the process limits the organic layer growth to a single monolayer, and FTIR measurements reveal that such monolayers are dense and well ordered. In contrast, hydrogen-terminated diamond sites remain unaffected by this process. This method is thus complementary to the UV-initiated reaction of alkenes with diamond, which exhibits the opposite reactivity contrast. Thermal alkylation increases the range of available diamond functionalization strategies and provides a means of straightforwardly forming single organic layers in order to engineer the surface properties of diamond.     

Structure-function analysis of an enzymatic prenyl transfer reaction identifies a reaction chamber with modifiable specificity

Fungal indole prenyltransferases participate in a multitude of biosynthetic pathways. Their ability to prenylate diverse substrates has attracted interest for potential use in chemoenzymatic synthesis. The fungal indole prenyltransferase FtmPT1 catalyzes the prenylation of brevianamide F in the biosynthesis of fumitremorgin-type alkaloids, which show diverse pharmacological activities and are promising candidates for the development of antitumor agents. Here, we report crystal structures of unliganded Aspergillus fumigatus FtmPT1 as well as of a ternary complex of FtmPT1 bound to brevianamide F and an analogue of its isoprenoid substrate dimethylallyl diphosphate. FtmPT1 assumes a rare α/β-barrel fold, consisting of 10 circularly arranged β-strands surrounded by α-helices. Catalysis is performed in a hydrophobic reaction chamber at the center of the barrel. In combination with mutagenesis experiments, our analysis of the liganded and unliganded structures provides insight into the mechanism of catalysis and the determinants of regiospecificity. Sequence conservation of key features indicates that all fungal indole prenyltransferases possess similar active site architectures. However, while the dimethylallyl diphosphate binding site is strictly conserved in these enzymes, subtle changes in the reaction chamber likely allow for the accommodation of diverse aromatic substrates for prenylation. In support of this concept, we were able to redirect the regioselectivity of FtmPT1 by a single mutation of glycine 115 to threonine. This finding provides support for a potential use of fungal indole prenyltransferases as modifiable bioreactors that can be engineered to catalyze highly specific prenyl transfer reactions.     

Circular dichroism in drug discovery and development: an abridged review

Chirality plays a fundamental role in determining the pharmacodynamic and pharmacokinetic properties of drugs, and contributes significantly to our understanding of the mechanisms that lie behind biorecognition phenomena. Circular dichroism spectroscopy is the technique of choice for determining the stereochemistry of chiral drugs and proteins, and for monitoring and characterizing molecular recognition phenomena in solution. The role of chirality in our understanding of recognition phenomena at the molecular level is discussed here via several selected systems of interest in the drug discovery and development area. The examples were selected in order to underline the utility of circular dichroism in emerging studies of protein–protein interactions in biological context. In particular, the following aspects are discussed here: the relationship between stereochemistry and pharmacological activity—stereochemical characterization of new leads and drugs; stereoselective binding of leads and drugs to target proteins—the binding of drugs to serum albumins; conformational transitions of peptides and proteins of physiological relevance, and the stereochemical characterization of therapeutic peptides.     

A Process to Produce Penicillin G Acylase by Surface-Adhesion Fermentation Using <Emphasis Type="Italic">Mucor griseocyanus</Emphasis> to Obtain 6-Aminopenicillanic Acid by Penicillin G Hydrolysis

The production of extracellular and mycelia-associated penicillin G acylase (maPGA) with Mucor griseocyanus H/55.1.1 by surface-adhesion fermentation using Opuntia imbricata, a cactus, as a natural immobilization support was studied. Enzyme activity to form 6-aminopencillanic acid (6-APA) from penicillin G was assayed spectrophotometrically. The penicillin G hydrolysis to 6-APA was evaluated at six different times using PGA samples recovered from the skim milk medium at five different incubation times. Additionally, the effect of varying the penicillin G substrate concentration level on the PGA enzyme activity was also studied. The maximum reaction rate, V _max, and the Michaelis constant, K _M, were determined using the Michaelis–Menten model. The maximum levels for maPGA and extracellular activity were found to be 2,126.50 international unit per liter (IU/l; equal to 997.83 IU/g of support) at 48 h and 755.33 IU/l at 60 h, respectively. Kinetics of biomass production for total biomass showed a maximum growth at 60 h of 3.36 and 2.55 g/l (equal to 0.012 g of biomass per gram of support) for the immobilized M. griseocyanus biomass. The maPGA was employed for the hydrolysis of penicillin G to obtain 6-APA in a batch reactor. The highest quantity of 6-APA obtained was 226.16 mg/l after 40-min reaction. The effect of substrate concentration on maPGA activity was evaluated at different concentrations of penicillin G (0–10 mM). K _M and V _max were determined to be 3.0 × 10⁻³ M and 4.4 × 10⁻³ mM/min, respectively.     

Global QSAR models of skin sensitisers for regulatory purposes

Background

The new European Regulation on chemical safety, REACH, (Registration, Evaluation, Authorisation and Restriction of CHemical substances), is in the process of being implemented. Many chemicals used in industry require additional testing to comply with the REACH regulations. At the same time EU member states are attempting to reduce the number of animals used in experiments under the 3 Rs policy, (refining, reducing, and replacing the use of animals in laboratory procedures). Computational techniques such as QSAR have the potential to offer an alternative for generating REACH data. The FP6 project CAESAR was aimed at developing QSAR models for 5 key toxicological endpoints of which skin sensitisation was one.

Results

This paper reports the development of two global QSAR models using two different computational approaches, which contribute to the hybrid model freely available online.

Conclusions

The QSAR models for assessing skin sensitisation have been developed and tested under stringent quality criteria to fulfil the principles laid down by the OECD. The final models, accessible from CAESAR website, offer a robust and reliable method of assessing skin sensitisation for regulatory use.

     

AuPt core–shell nanocatalysts with bulk Pt activity

In the presented work, the evaluation of an unsupported AuPt core–shell catalyst for the oxygen reduction reaction is introduced. Applying only basic chemicals in an upscalable synthesis route, it is demonstrated that uniform, flat, and complete Pt layers around a spherical Au core are obtained. The electrocatalytic measurements show that the surface area specific activity of the AuPt core–shell catalyst towards the important oxygen reduction reaction equals the one of polycrystalline bulk Pt. To our knowledge, this is the first time that the unfavorable particle size effect of Pt nanoparticles could be by-passed for a nanoscale catalyst.     

The Role of Ligand Topology in the Decomplexation of Luminescent Lanthanide Complexes by Dipicolinic Acid

In this study, we present the aqueous solution behavior of two luminescent lanthanide antenna complexes (Eu³⁺? 1 , Dy³⁺? 9 ) with different ligand topologies in the presence of dipicolinic acid (DPA, pyridine‐2,6‐dicarboxylic acid). Macrocyclic (1,4,7,10‐tetraazacyclododecane‐1,4,7‐triacetic acid, DO3A, 9 ) and acyclic (1,4,7‐triazaheptane‐1,1,7,7‐tetraacetic acid, DTTA, 1 ) ligands have been selected to form a ratiometric pair in which Dy³⁺? 9 acts as a reference and Eu³⁺? 1 acts as a probe for the recognition of DPA. The pair of luminescent complexes in water reveals the capability to work as a DPA luminescent sensor. The change of emission intensity of Eu³⁺ indicates the occurrence of a new sensitization path for the lanthanide cation through excitation of DPA. NMR evidence implies the presence of free 1 and mass spectrometry shows the formation of emitting [EuDPA₂]^? as a result of a ligand exchange reaction.     

The Evolving Contribution of Mass Spectrometry to Integrative Structural Biology

Protein complexes are key catalysts and regulators for the majority of cellular processes. Unveiling their assembly and structure is essential to understanding their function and mechanism of action. Although conventional structural techniques such as X-ray crystallography and NMR have solved the structure of important protein complexes, they cannot consistently deal with dynamic and heterogeneous assemblies, limiting their applications to small scale experiments. A novel methodological paradigm, integrative structural biology, aims at overcoming such limitations by combining complementary data sources into a comprehensive structural model. Recent applications have shown that a range of mass spectrometry (MS) techniques are able to generate interaction and spatial restraints (cross-linking MS) information on native complexes or to study the stoichiometry and connectivity of entire assemblies (native MS) rapidly, reliably, and from small amounts of substrate. Although these techniques by themselves do not solve structures, they do provide invaluable structural information and are thus ideally suited to contribute to integrative modeling efforts. The group of Brian Chait has made seminal contributions in the use of mass spectrometric techniques to study protein complexes. In this perspective, we honor the contributions of the Chait group and discuss concepts and milestones of integrative structural biology. We also review recent examples of integration of structural MS techniques with an emphasis on cross-linking MS. We then speculate on future MS applications that would unravel the dynamic nature of protein complexes upon diverse cellular states.

Open image in new window

Graphical Abstract ?