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Groves P.,Institute Tecnologia Quimica e Biologica ITQB UNL | Groves P.,CSIC - Biological Research Center | Strzelecka-Kiliszek A.,Nencki Institute of Experimental Biology | Sekrecka-Belniak A.,Nencki Institute of Experimental Biology | And 6 more authors.
Organic and Biomolecular Chemistry

Fluorescent analogues provide important tools for biochemical/biophysical research. However, the analogues contain chemical modifications much larger than those known to affect ligand-binding, such as the inversion of a carbon centre or substitution of an atom. We lack experimental tools and protocols to select the most appropriate fluorescent analogue. Herein, we use several NMR spectroscopy methods, including Saturation Transfer Difference (STD), STD competition and transferred nuclear Overhauser effect spectroscopy (Tr-NOESY), as tools to select appropriate fluorescent probes. Annexin A6 (AnxA6) is a ubiquitous protein that forms in vitro GTP-induced ion channels. We used this protein as a model and screened guanosine triphosphate (GTP) and four fluorescent analogues against AnxA6. STD reported that the GTP moiety of all ligands made similar contacts with the protein, despite additional interactions between the fluorescent tags and AnxA6. Competition STD experiments verified that the analogues and GTP bind to the same site. Tr-NOESY indicated that the bound conformation of the base relative to ribose is altered for some analogues compared to GTP. MANT-GTP or the BODIPY thioester of guanosine 5′-O-(3-thiotriphosphate) are the most suitable fluorescent analogues for AnxA6, according to NMR. These results reveal NMR as a useful technique to select and design proper fluorescent tags for biochemical/biophysical assays.© 2013 The Royal Society of Chemistry. Source

Mendes M.D.,University of Lisbon | Figueiredo A.C.,University of Lisbon | Margarida Oliveira M.,Institute Tecnologia Quimica e Biologica ITQB UNL | Trindade H.,University of Lisbon
Plant Cell, Tissue and Organ Culture

Thymus caespititius Brot. is an important aromatic species, due to synthesis and production of essential oils for the pharmaceutical and food industries. In the present study, levels of essential oils from two chemotypes, including carvacrol/thymol (CT) and sabinene/carvacrol (SC), were evaluated in proliferating shoot cultures (6-12 subcultures following establishment) and compared to those from field-grown plants. The essential oils were isolated by hydrodistillation and analysed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). Cultures grown under in vitro culture conditions, evaluated over six subcultures, were found to maintain stable composition of essential oils. For the CT chemotype, carvacrol (42 %) and thymol (23 %) were the main essential oil components detected in field-grown plants; in proliferating shoot cultures the levels detected attained 17-25 % in the case of carvacrol and 18-23 % in that of thymol, closely followed by carvacryl acetate (15-23 %) and thymyl acetate (11-15 %). For the SC chemotype, carvacrol (13-28 %), sabinene (18-45 %), and thymol (9-12 %) were the main essential oil components detected in both field-grown and proliferating shoot cultures. Our experiments showed that the essential oil composition in proliferating shoot cultures was not only stable, but also qualitatively similar to that of field-grown plants, notwithstanding minor quantitative differences. © 2012 Springer Science+Business Media Dordrecht. Source

Zanol G.C.,Instituto Nacional Of Recursos Biologicos | Cunha J.,Instituto Nacional Of Recursos Biologicos | Brazao J.S.,Instituto Nacional Of Recursos Biologicos | Fevereiro P.S.,Institute Tecnologia Quimica e Biologica ITQB UNL | Eiras-Dias J.E.,Instituto Nacional Of Recursos Biologicos
Acta Horticulturae

Many mutations affecting berry colour in Vitis vinifera cultivars have been reported. In the Portuguese National Ampelographic Collection at INIA - Dois Portos, an accession was found that exhibits, in some vines and at whole plant level, completely white-skin bunches. Other vines exhibit branches giving rise to only white-skin berries and branches which present, in the same bunch, three different coloured berries (completely blue black or white berries and berries showing longitudinally white or blue black sectors). Genomic DNA was extracted from leaves and berries of all phenotypes and analyzed at six nuclear SSR (Simple Sequence Repeat) loci. The SSR analysis revealed that the fragments amplified (from all plants presenting bud sports) coincided in size with those from the Portuguese blue black-berried cultivar 'Alfrocheiro Preto'. The cultivar ancestry was further confirmed with ampelometric descriptors (OIV, 2007) and phenological observations. We infer that the whiteberried plants are a bud sport arisen in 'Alfrocheiro Preto' through a somatic mutation. Source

Ma P.,University of Leeds | Ma P.,Institute Tecnologia Quimica e Biologica ITQB UNL | Nishiguchi K.,Kyushu University | Yuille H.M.,University of Leeds | And 3 more authors.
FEBS Letters

Siamycin I disrupts growth and quorum sensing in Enterococcus faecalis. Using purified intact protein, we demonstrate here that quorum membrane sensor kinase FsrC is a direct target of siamycin I, reducing pheromone-stimulated autophosphorylation activity by up to 91%. Inhibition was non-competitive with ATP as substrate. Other ATP-binding enzymes were also inhibited, including nine other membrane sensor kinases of E. faecalis, Rhodobacter sphaeroides PrrB, porcine Na+-dependent ATPase and the catalytic subunit of bovine protein kinase A, but not bacterial β-galactosidase, confirming targeted inhibition of a wide range of ATP dependent reactions, and elucidating a likely mechanism underlying the lethality of the inhibitor. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Source

De La Gandara M.P.,Rockefeller University | Garay J.A.R.,Pennsylvania State University | Mwangi M.,Pennsylvania State University | Tobin J.N.,Rockefeller University | And 17 more authors.
Journal of Clinical Microbiology

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillinresistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most-46 of the 63-wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL+) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing. Copyright © 2015, American Society for Microbiology. All Rights Reserved. Source

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