Growth hormone improves lipoprotein concentration and arylesterase activity in mice with an atherogenic lipid profile induced by lactalbumin

E. LOPEZ-OLIVA, M. NUS, A. AGIS-TORRES, W. VILLARO, J.M. SANCHEZ-MONTERO, E. MUÑOZ-MARTINEZ, F.J. SANCHEZ-MUÑIZ

Sección Departamental de Fisiología Animal, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain

Departamento de Nutrición y Bromatología I (Nutrición), Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain

Biotransformations Group, Departamento de Química Orgánica y Farmacéutica, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain

The effect of growth hormone (GH) on arylesterase (AE), one of the activities of paraoxonase, has never been studied. The aims of the present study in mice were: (a) to compare the effect of age and sex on serum lipid and lipoprotein levels after consumption of lactalbumin- v. chow-based diets and (b) to study the effect of GH administration, age and sex on serum AE activity, lipid and lipoprotein and body fat levels in mice fed a lactalbumin diet. Seventy-two mice were divided into three age- and sex-matched experimental groups: (1) control chow (CC), (2) non-GH lactalbumin (NGL) and (3) GH-treated lactalbumin (GL) mice. Lactalbumin increased total cholesterol, (LDL+VLDL)-cholesterol and TAG and diminished HDL-cholesterol in all animals

http://dx.doi.org/10.1017/S0007114508025014

A global benchmark study using affinity-based biosensors

Prof. MJ Hernaiz is one of the co-authors of this global study

To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
Keywords: Biacore; Kinetics; Optical biosensor; Surface plasmon resonance

http://dx.doi.org/10.1016/j.ab.2008.11.021

Glycan Tagging to Produce Bioactive Ligands for a Surface Plasmon Resonance (SPR) Study via Immobilization on Different Surfaces.

F.J MUÑOZ, J. PEREZ, A. RUMBERO, J.I. SANTOS, F.J. CAÑADA, S. ANDRE, H.J. GABIUS, J. JIMENEZ-BARBAERO, J.V. SINISTERRA, M.J. HERNAIZ.

Departamento de Quimica Organica y Farmaceutica, Universidad Complutense de Madrid, Plaza Ramon y Cajal s/n. 28040 Madrid, Spain
Departamento de Quimica Organica, Universidad Autonoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain

Departamento de Ciencia de Proteinas, CIB-CSIC, c/Ramiro de Maeztu 9, 28040 Madrid, Spain, Institut fur Physiologische Chemie, Tierarztliche Fakultat, Ludwig-Maximilians-Universitat, Munchen, Veterinarstr 13, 80539 Munchen, Germany

Servicio de Biotransformaciones Industriales, Parque Cientifico de Madrid C/Santiago Grisolia, 28760 Tres Cantos, Spain & Servicio de Interacciones Biomoleculares, Parque Cientifico de Madrid, Pz/Ramon y Cajal s/n. 28040 Madrid, Spain.

Suitable glycan derivatives will find immediate application in the study of their interactions. Here, we present an efficient synthetic strategy to introduce a fluorescent tag functionalized with an amino group into a model disaccharide structure (lactose). This strategy led to the maintenance of bioactivity, checked by the study of the interaction of this bioconjugate with a plant lectin (mistletoe lectin 1) by NMR spectroscopy, computational docking, and surface plasmon resonance (SPR). To demonstrate the versatility of this approach, we immobilized the new glycan derivatives on different surfaces, and a comparative analysis is presented and can be successfully used for biomimetic carbohydrate-protein interaction studies on the SPR biochip.

http://dx.doi.org/10.1021/bc800350q

Biotransformations. vol. 1, pp. 212-251. Encyclopedia of Microbiology 3rd Edition. Oxford. Elsevier

J.D. CARBALLEIRA, J. FERNANDEZ-LUCAS, M.A. QUEZADA, M.J. HERNAIZ, A.R. ALCANTARA, Y. SIMEO, J.V. SINISTERRA

The application of whole-cell-catalyzed biotransformations is an emerging field that opens up a myriad of new possibilities for the industrial application of biocatalysts. In this article, the new technologies to design tailor-made cells for specific reactions as well as some interesting applied processes catalyzed by intracellular enzymes are presented.

Encyclopedia of Microbiology
Moselio Schaechter, San Diego State University, CA, USA
Six-Volume Set, 1-6 Hardbound, 4600 pages, publication date: APR-2009
ISBN-13: 978-0-12-373939-1
ISBN-10: 0-12-373939-X
Imprint: ACADEMIC PRESS

Monascus kaoliang CBS 302.78 immobilized in polyurethane foam using iso-propanol as co-substrate: Optimized immobilization conditions of a fungus as b

M.A. Quezada, J.D. Carballeira and J.V. Sinisterra

Department of Chemistry, Faculty of Chemical Engineering, University of Trujillo, Peru
Industrial Biotransformations Service, Scientific Park of Madrid, C/Santiago Grisolia No. 2, Parque Tecnológico de Madrid, 28760 Tres Cantos, Madrid, Spain

Biotransformations Group, Organic and Pharmaceutical Chemistry Department, Faculty of Pharmacy, Universidad Complutense, 28040 Madrid, Spain

Abstract
Monascus kaoliang was selected after a microbial screening as a highly active and selective whole cell catalyst for the reduction of ketones. In the present paper we describe the optimum growing conditions and an interesting immobilization procedure by adsorption in polyurethane foams (PUFs). This methodology is easy to perform and the immobilized catalyst is active, stable and reusable. The use of different co-substrates for cofactor regeneration was also tested and iso-propanol (i-PrOH) was found as the best co-substrate, as it leads to a catalyst reusable for 17 cycles, displaying better NADH regeneration properties than others e.g., glucose (10 cycles) or saccharose (6 cycles). The reduction of different prochiral ketones showed that the ketone reductase activity of this mould follows the Prelog’s rule and kinetic experiments demonstrated that the process follows a pseudo-first kinetic order.

Biotransformation of Terpenoids: A Green Alternative for Producing Molecules with Pharmacological Activity

Y. Simeó and J.V. Sinisterra

Mini Reviews in Organic Chemistry 2009

Terpenoids are natural products of great interest due to their broad application scope. They are employed as agrochemicals, drugs, fragrances, flavours and pigments. In the search of new derivatives with improved properties, the use of biocatalysts is being constantly increased, especially in redox processes. They can give rise to stereo- and regioselective products and/or compounds functionalized in remote positions difficult to reach by means of traditional organic chemistry. In this review, the application of whole cell catalyzed biotransformations of terpenoids to obtain new drug targets or to increase the pharmacological activity is presented.

http://www.bentham.org/mroc/

Defining the Epitope Region of a Peptide from the Streptomyces coelicolor Phosphoenolpyruvate:Sugar Phosphotransferase System Able to Bind to the Enzy

E. HURTADO-GOMEZ, O. ABIAN, F. J. MUÑOZ , M.J. HERNAIZ, A. VELAZQUEZ-CAMPOY, J.L. NEIRA

Abstract
The bacterial PEP:sugar PTS consists of a cascade of several proteins involved in the uptake and phosphorylation of carbohydrates, and in signal transduction pathways. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments able to interfere with the first reaction of the protein cascade: the phosphorylation of the HPr by the first enzyme, the so-called enzyme EI. To that end, we designed a peptide, HPr9–30, spanning residues 9 to 30 of the intact HPr protein, containing the active site histidine (His-15) and the first α-helix of HPr of Streptomyces coelicolor, HPrsc. By using fluorescence and circular dichroism, we first determined qualitatively that HPrsc and HPr9–30 did bind to EIsc, the enzyme EI from S. coelicolor. Then, we determined quantitatively the binding affinities of HPr9–30 and HPrsc for EIsc by using ITC and STD-NMR. The STD-NMR experiments indicate that the epitope region of HPr9–30 was formed by residues Leu-14, His-15, Ile-21, and Val-23. The binding reaction between EIsc and HPrsc is enthalpy driven and in other species is entropy driven; further, the affinity of HPrsc for EIsc was smaller than in other species. However, the affinity of HPr9–30 for EIsc was only moderately lower than that of EIsc for HPrsc, suggesting that this peptide could be considered a promising hit compound for designing new inhibitors against the PTS.
Abbreviations: PEP, phosphoenolpyruvate; CD, circular dichroism; EIsc, enzyme I of Streptomyces coelicolor; EIec, enzyme I of E. coli; HPrec, histidine phosphocarrier protein of Escherichia coli; HPrsc, histidine phosphocarrier protein of Streptomyces coelicolor; HPr9-30, peptide comprising residues Gly-9 to Gly-30 of the intact HPrsc; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; PTS, sugar phosphotransferase system; STD, saturation transfer difference; TFE, trifluoroethanol; TSP, sodium trimethylsilyl [2,2,3,3-2H4] propionate; UV, ultraviolet

http://dx.doi.org/10.1529/biophysj.107.126664

Versatile strategy for the synthesis of biotin-labelled glycans, their immobilization to establish a bioactive surface and interaction studies with a

F.J MUÑOZ, A. RUMBERO, J.V. SINISTERRA, J.I. SANTOS, S. ANDRE, H.J. GABIUS, J. JIMENEZ-BARBERO, M.J. HERNAIZ

Abstract The emerging role of glycans as versatile biochemical signals in diverse aspects of cellular sociology calls for establishment of sensitive methods to monitor carbohydrate recognition by receptors such as lectins. Most of these techniques involve the immobilization of one of the binding partners on a surface, e.g. atomic force microscopy, glycan array and Surface Plasmon Resonance (SPR), hereby simulating cell surface presentation. Here, we report the synthesis of fluorescent glycoconjugates, with a functionalization strategy which avoids the frequently occurring ring opening at the reducing end for further immobilization on a surface or derivatization with biotin. In order to improve the versatility of these derivatized glycans for biological studies, a new approach for the synthesis of biotinylated and fluorescent glycans has also been realized. Finally, to illustrate their usefulness the neoglycoconjugates were immobilized on different surfaces, and the interaction analysis with a model lectin, the toxin from mistletoe, proved them to act as potent ligands, underscoring the merit of the presented synthetic approach.
Electronic supplementary material The online version of this article (doi:10.1007/s10719-008-9115-y) contains supplementary material, which is available to authorized users.

http://dx.doi.org/10.1007/s10719-008-9115-y

Esterification reactions catalyzed by lipases immobilized in organogels: effect of temperature and substrate diffusion

M. ZOUMPANIOTI, P. PARMAKLIS, P. DOMINGUEZ DE MARIA, H. STAMATIS, J.V. SINISTERRA, A. XENAKIS

Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece

Biotransformations Group, Faculty of Pharmacy, Universidad Complutense, Madrid, Spain

Biological Applications and Technologies Department, University of Ioannina, Ioannina, Greece

Abstract

Rhizomucor miehei lipase was immobilized in hydroxy(propylmethyl) cellulose or agar gels containing lecithin or AOT microemulsions. The effect of the diffusion of substrates and products to this catalyst was studied, as well as the effect of temperature on the initial rate of ester synthesis. The composition of the gel affects the reaction rate due to mass transport phenomena. The apparent activation energies were higher for the systems based on agar, independently of the microemulsion used, and lower for the systems based on AOT microemulsions, independently of the polymer used.

http://dx.doi.org/10.1007/s10529-008-9734-1