Stabilisation of mixed peptide/lipid complexes in selective antifungal hexapeptides

AbstractThe design of antimicrobial peptides could have benefited from structural studies of known peptides having specific activity against target microbes, but not toward other microorganisms. We have previously reported the identification of a series of peptides (PAF-series) active against certain postharvest fungal phytopathogens, and devoid of toxicity towards E. coli and S. cerevisiae [López-Garcı́a et al. Appl. Environ. Microbiol. 68 (2002) 2453]. The peptides inhibited the conidia germination and hyphal growth. Here, we present a comparative structural characterisation of selected PAF peptides obtained by single-amino-acid replacement, which differ in biological activity. The peptides were characterised in solution using fluorescence and circular dichroism (CD) spectroscopies. Membrane and membrane mimetic–peptide interactions and the lipid-bound structures were studied using fluorescence with the aid of extrinsic fluorescent probes that allowed the identification of mixed peptide/lipid complexes. A direct correlation was found between the capability of complex formation and antifungal activity. These studies provide a putative structural basis for the mechanism of action of selective antifungal peptides

A fluorescent polarization-based assay for the identification of disruptors of the RCAN1/calcineurin A protein complex

5 pages, 4 figures, a table. 19891949 [PubMed]Calcineurin is a Ca(2+)/calmodulin-dependent serine/threonine protein phosphatase involved in many biological processes and developmental programs, including immune response. One of the most studied substrates of calcineurin is the transcription factor NFAT (nuclear factor of activated T cells) responsible for T-cell activation. Different anticalcineurin drugs, such as cyclosporine A and FK506, are the most commonly used immunosuppressants in transplantation therapies. Unfortunately, their mechanism of action, completely blocking the calcineurin phosphatase activity while also requiring continuous administration, bears severe side effects. During recent years, the family of regulators of calcineurin (RCAN) has been described and studied extensively as modulators of calcineurin signaling pathways. The RCAN1 region, spanning amino acids 198 to 218 and responsible for inhibiting the calcineurin-NFAT signaling pathway in vivo, has been identified. An RCAN1-derived peptide spanning this sequence interferes with the calcineurin-NFAT interaction without affecting the general calcineurin phosphatase activity. Here we report the development of an optimized in vitro high-throughput fluorescence polarization assay based on the disruption of the RCAN1(198-218)-CnA interaction for identifying molecules with immunosuppressant potential. This approach led us to identify dipyridamole as a disruptor of such interaction. Moreover, three small molecules with a potential immunosuppressive effect were also identifiedThis work was supported by grants from Fundació La Marató de TV3 (Ref. 030830), the Spanish Ministry of Education and Science (SAF2006-04815, BIO2004-00998, BIO2007-60066, CTQ2005-00995/BQU), the Fundación Mutua Madrileña 2007 and from the Generalitat de Catalunya (Ref. 2006 BE 00051)Peer reviewe

Vareniclina. Un paso más en la lucha contra el tabaquismo

Recientemente la Food and Drugs Administration ha aprobado Vareniclina, fármaco para la deshabituación tabáquica que presenta un novedoso mecanismo de acción, agonismo parcial de receptores nicotínicos de acetilcolina a4ß2.Hemos revisado la farmacología, eficacia y seguridad de Vareniclina y su utilidad en los procesos de deshabituación, usando como fuentes bibliográficas la base de datos MEDLINE , MD consults, American journal of Addictions y distintos manuales de referencia.De los tres ensayos clínicos publicados, dos de ellos, Jorenby y Gonzales comparan la eficacia y seguridad de Vareniclina 1mg/12h frente a Bupropion 150mg/12h y placebo, durante un periodo de 52 semanas, obteniéndose los siguentes resultados: 23% de abstinencia con Vareniclina, 14,6% con Bupropion y 10,3% con Placebo en el ensayo de Jorenby. Resultados similares obtuvo Gonzales: 21,9% de abstinencia con Vareniclina frente a 16,1% con Bupropion y 8,4% con placebo.El ensayo Tonstad determina que los pacientes que dejan de fumar tras 12 semanas con Vareniclina mantienen porcentajes elevados en las 12 semanas adicionales de tratamiento y hasta 52 semanas totales, obtuviéndose un 49,6% de abstinencia con Vareniclina frente a 36,9% con placebo.Con los estudios publicados a día de hoy, podemos concluir que Vareniclina en la dosis habitual (1mg/12h) muestra mayor eficacia a corto y largo plazo tanto frente a placebo cómo frente a tratamientos habituales en la deshabituación tabáquica, con un buen perfil de seguridad y bajo porcentaje de reacciones adversas

Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion

Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target mem- brane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all D-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell-cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences

Influence of hydrophobic matching on association of model transmembrane fragments containing a minimised glycophorin A dimerisation motif

The principles that govern the folding and packing of membrane proteins are still not completely understood. In the present work, we have revisited the glycophorin A (GpA) dimer- isation motif that mediates transmembrane (TM) helix associa- tion, one of the best-suited models of membrane protein oligomerisation. By using artificial polyleucine TM segments we have demonstrated in this study that a pattern of only five amino acids (GVxxGVxxT) promotes specific dimerisation. Fur- ther, we have used this minimised GpA motif to assess the influ- ence of hydrophobic matching on the TM helix packing process in detergent micelles and found that this factor modulates helix-helix association and/or dissociation between TM fragments

Influence of proline residues in transmembrane helix packing

Integral membrane proteins often contain proline residues in their alpha-helical transmembrane (TM) fragments, which may strongly influence their folding and association. Pro-scanning mutagenesis of the helical domain of glycophorin A (GpA) showed that replacement of the residues located at the center abrogates helix packing while substitution of the residues forming the ending helical turns allows dimer formation. Synthetic TM peptides revealed that a point mutation of one of the residues of the dimerization motif (L75P) located at the N-terminal helical turn of the GpA TM fragment, adopts a secondary structure and oligomeric state similar to the wild-type sequence in detergents. In addition, both glycosylation mapping in biological membranes and molecular dynamics showed that the presence of a proline residue at the lipid/water interface has as an effect the extension of the helical end. Thus, helix packing can be an important factor that determines appearance of proline in TM helices. Membrane proteins might accumulate proline residues at the two ends of their TM segments in order to modulate the exposition of key amino acid residues at the interface for molecular recognition events while allowing stable association and native folding

Transcendencia del cocaetileno en el consumo combinado de etanol y cocaína

Cada vez es más frecuente encontrar a nivel asistencial las repercusiones del consumo abusivo de cocaína y etanol.La interacción metabólica del etanol y la cocaína da lugar a un nuevo metabolito denominado cocaetileno, el cual juega un papel importante en el aumento del placer y la toxicidad derivada del consumo de ambas drogas. Este produce un aumento considerable de la euforia y duración de la misma, así como una disminución de la disforia por abstinencia a la cocaína, unido a consumos más compulsivos.Todo esto influye en una mayor pérdida del control del consumo, más problemas sociales y conductas violentas y de riesgo.Gran parte de las diferencias observadas en la acción de ambas sustancias en su administración conjunta, pueden ser explicadas por las modificaciones en la farmacocinética de dichas drogas y por la potencial acción tóxica del cocaetileno, la cual se suma a las respectivas acciones del etanol y la cocaína, pasando a ser la base de los cuadros clínicos de mayor gravedad observados en este tipo de adicción.El objetivo de esta revisión es recoger los efectos psicológicos y somáticos del consumo combinado del etanol y la cocaína, centrándonos en la acción tóxica del cocaetileno sobre el organismo, mediante la revisión de la bibliografía primaria obtenida a través de la base de datos Medline (PubMed 1979-2006), en Índice Médico Español (IME), en datos de la Organización Mundial de la Salud (O.M.S), Revista del Instituto de Investigación de Drogodependencias, datos del Plan Nacional sobre Drogas (PNSD), revista de Adicciones, así como de diversas monografías relacionadas.La compresión de los efectos derivados del consumo de etanol y cocaína así como los mecanismos implicados en la toxicidad directa del cocaetileno, pueden ayudar a resolver los problemas tanto físicos como psicológicos ocasionados en este tipo de toxicomanía

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in β-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future

Analyse der Arzneimitteltherapie von Rheumapatienten im Erwachsenenalter : das Versorgungsgeschehen im Lichte der Leitlinienempfehlungen

The aim of this dissertation is to demonstrate the further developments in the provision of rheumatological health care with particular focus on evidence-based guideline recommendations. One of the issues to be examined is to identify gaps - defined as conditions of insufficient medical care between practical daily care and (theoretical) guideline recommendations differentiating various aspects of care. Are there still references about a lack of provison of medical care or is it possible to reveal further developments that can be assessed as positive respectively? Avenues for future action are to be pointed out against the background of these findings with regard to measures which have already been established as well as to aspired solutions in order to optimate the provision of rheumatological health care. A further glimpse of the future is disclosed by taking up the biosimilar-issue: As many bio-pharmaceuticals lose patent protection in coming years, biosimilar products are expected to play a key role in controlling pharmaceutical expenditure - while maintaining consistent quality of medical care. Assessing opportunities and challenges of these new medical products will round off this dissertation