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1.
Antimicrob Agents Chemother ; 56(4): 1749-55, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22232283

RESUMEN

Rhodococcus equi, the causal agent of rhodococcosis, is a severe pathogen of foals but also of immunodeficient humans, causing bronchopneumonia. The pathogen is often found together with Klebsiella pneumoniae or Streptococcus zooepidemicus in foals. Of great concern is the fact that some R. equi strains are already resistant to commonly used antibiotics. In the present study, we evaluated the in vitro potential of two equine antimicrobial peptides (AMPs), eCATH1 and DEFA1, as new drugs against R. equi and its associated pathogens. The peptides led to growth inhibition and death of R. equi and S. zooepidemicus at low micromolar concentrations. Moreover, eCATH1 was able to inhibit growth of K. pneumoniae. Both peptides caused rapid disruption of the R. equi membrane, leading to cell lysis. Interestingly, eCATH1 had a synergic effect together with rifampin. Furthermore, eCATH1 was not cytotoxic against mammalian cells at bacteriolytic concentrations and maintained its high killing activity even at physiological salt concentrations. Our data suggest that equine AMPs, especially eCATH1, may be promising candidates for alternative drugs to control R. equi in mono- and coinfections.


Asunto(s)
Infecciones por Actinomycetales/tratamiento farmacológico , Infecciones por Actinomycetales/microbiología , Antibacterianos/farmacología , Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/microbiología , Rhodococcus equi , alfa-Defensinas/farmacología , Infecciones por Actinomycetales/veterinaria , Animales , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Dicroismo Circular , Farmacorresistencia Bacteriana , Sinergismo Farmacológico , Femenino , Hemólisis , Caballos , Liposomas/química , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Rastreo , Fosfolípidos/química , Rhodococcus equi/efectos de los fármacos , Rhodococcus equi/ultraestructura , Tolerancia a la Sal , Ovinos , Células Vero , alfa-Defensinas/química
2.
Bioorg Med Chem Lett ; 20(18): 5541-3, 2010 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-20692829

RESUMEN

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei.


Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Plasmodium falciparum/enzimología , Ácidos Sulfínicos/química , Ácidos Sulfínicos/farmacología , Trypanosoma brucei brucei/enzimología , Animales , Cisteína Endopeptidasas/metabolismo , Disulfuros , Ajo/química , Humanos , Malaria Falciparum/tratamiento farmacológico , Plasmodium falciparum/efectos de los fármacos , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico
3.
J Med Chem ; 52(18): 5662-72, 2009 Sep 24.
Artículo en Inglés | MEDLINE | ID: mdl-19715342

RESUMEN

A new class of cysteine protease inhibitors based on fumaric acid derived oligopeptides was successfully identified from a high-throughput screening of a solid-phase bound combinatorial library. As target enzymes falcipain and rhodesain were used, which play important roles in the life cycles of the parasites which cause malaria (Plasmodium falciparum) and African sleeping sickness (Trypanosoma brucei rhodesiense). The best inhibitors with unusual amino acid sequences not reported before for this type of enzyme were also fully analyzed in detail in solution. K(i) values in the lower micromolar and even nanomolar region were found. Some inhibitors are even active against plasmodia and show good selectivity relative to other enzymes. Also the mechanism of action was studied and could be shown to be irreversible inhibition.


Asunto(s)
Técnicas Químicas Combinatorias , Cisteína Endopeptidasas/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Fumaratos/química , Fumaratos/farmacología , Biblioteca de Péptidos , Plasmodium falciparum/enzimología , Secuencia de Aminoácidos , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Antiprotozoarios/farmacología , Catepsina L , Catepsinas/química , Bovinos , Cisteína Endopeptidasas/química , Inhibidores de Cisteína Proteinasa/síntesis química , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Diamida/química , Fumaratos/síntesis química , Humanos
4.
Proteomics ; 9(13): 3507-21, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19609960

RESUMEN

Over the last decade, an increasing prevalence of peanut allergies was observed worldwide. Peanuts are meanwhile categorized among the most dangerous food allergens. This is particularly relevant since peanut-derived ingredients are widely used in industrial food production. To minimize the problem of hidden food allergens causing severe anaphylactic reactions, pre-packaged food containing peanut components needs to be classified according to European ruling since 2005. Food companies search for strategies to reduce the allergenicity of peanut-derived food additives either by genetically altering the allergen content or by identifying peanut varieties with low levels of major allergens. In our study, we focused on peanut extracts from Indonesia that apparently contain lower levels of the major Arachis hypogaea allergen 1 (Ara h 1). Basic extracts of Virginia-type and Indonesian peanuts were compared by 1- and 2-DE. We identified more than hundred individual components in these extracts by MS and provide a high-resolution allergen map that also includes so far unknown fragments of major peanut allergens. The reduced level of Ara h 1 associated with a significantly lower abundance of the most potent peanut allergen Ara h 2 in various Indonesian peanuts was also confirmed by Western blotting with monoclonal antibodies and sera of allergic patients.


Asunto(s)
Antígenos de Plantas/análisis , Arachis/química , Glicoproteínas/análisis , Extractos Vegetales/química , Proteínas de Plantas/análisis , Proteoma/análisis , Anticuerpos Monoclonales/inmunología , Antígenos de Plantas/inmunología , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/inmunología , Humanos , Espectrometría de Masas , Extractos Vegetales/inmunología , Proteínas de Plantas/inmunología , Proteómica
5.
Vet Microbiol ; 136(3-4): 335-40, 2009 May 12.
Artículo en Inglés | MEDLINE | ID: mdl-19167844

RESUMEN

Human psoriasin (S100A7) has originally been described as a member of the family of S100 calcium-binding proteins which is overexpressed in patients suffering from psoriasis. The bovine homolog was first identified as a cow-derived respiratory allergen. As Escherichia coli mastitis is a common problem in dairy cattle, and human psoriasin was found to exhibit antimicrobial activity preferentially against E. coli, we examined whether the bovine mRNA is expressed in the mammary gland. To demonstrate the antimicrobial activity of bovine psoriasin, we isolated cDNA from the udder, cloned the bovine psoriasin gene in a bacterial expression vector, and the recombinant protein was expressed in BL21 cells. The in vitro antibacterial activity was tested by performing microdilution susceptibility tests and radial diffusion assays with eight different bacterial strains, thereof three different E. coli strains, and one yeast. The antimicrobial activity of the recombinant bovine psoriasin is comparable with human psoriasin and also limited to E. coli. Psoriasin appears to be a part of the local host defense mechanism in the udder, is a putative candidate for a cow-specific factor influencing mastitis susceptibility, and a possible alternative to conventional antibiotics.


Asunto(s)
Proteínas de Unión al Calcio/farmacología , Pruebas de Sensibilidad Microbiana/veterinaria , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Bovinos , Cromatografía en Gel/veterinaria , Dicroismo Circular/veterinaria , Clonación Molecular , ADN Complementario/genética , Femenino , Humanos , Datos de Secuencia Molecular , ARN/química , ARN/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100 , Alineación de Secuencia
6.
Biol Chem ; 389(12): 1523-5, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18844447

RESUMEN

A range of various assays to measure chemosusceptibility of Plasmodium falciparum have been described in the literature. As the screening of a plethora of compounds for antiplasmodial activity is urgently needed and becomes a constantly increasing routine analysis, a test system has to fulfill the following requirements: sensitivity, reliability, simplicity of performance, high-throughput compatibility, and cost-effectiveness. Here, we describe an assay that fulfills all criteria and in which the fluorescent SYTOX Green dye is introduced to determine growth inhibition of Plasmodia in in vitro cultures.


Asunto(s)
Antimaláricos/farmacología , Evaluación Preclínica de Medicamentos/economía , Evaluación Preclínica de Medicamentos/métodos , Eritrocitos/parasitología , Plasmodium falciparum/fisiología , Animales , Cloroquina/farmacología , Colorantes Fluorescentes , Humanos , L-Lactato Deshidrogenasa/metabolismo , Compuestos Orgánicos , Plasmodium falciparum/efectos de los fármacos , Espectrometría de Fluorescencia
7.
J Immunol ; 181(2): 1083-95, 2008 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-18606660

RESUMEN

Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair.


Asunto(s)
Aeromonas/inmunología , Péptidos Catiónicos Antimicrobianos/biosíntesis , Bacterias Grampositivas/inmunología , Hirudo medicinalis/fisiología , Microglía/metabolismo , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Axotomía , Secuencia de Bases , Sistema Nervioso Central/citología , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiología , Exocitosis , Hirudo medicinalis/genética , Hirudo medicinalis/inmunología , Hirudo medicinalis/microbiología , Microglía/citología , Microglía/inmunología , Datos de Secuencia Molecular , Regeneración Nerviosa , Neuronas/citología , Neuronas/inmunología , Alineación de Secuencia
8.
J Biol Chem ; 279(25): 25955-8, 2004 Jun 18.
Artículo en Inglés | MEDLINE | ID: mdl-15075336

RESUMEN

The pore-forming polypeptides of Naegleria fowleri, naegleriapores A and B, are processed from separate multipeptide precursor structures. According to their transcripts, each precursor molecule appears to contain additional naegleriapore-like polypeptides, all of which share a structural motif of six invariant cysteine residues within their amino acid sequence. To identify the putative pronaegleriapore-derived peptides at the protein level, amoebic extracts were screened for small cysteine-rich polypeptides by fluorescently labeling their cysteine residues. Three novel naegleriapore isoforms derived from the precursor molecule of naegleriapore B were identified. Two of the isoforms were purified to homogeneity and tested for their biological activity. The pore-forming activity of the novel peptides was remarkably lower than that of the originally isolated naegleriapores, but both peptides killed bacteria by permeabilizing their cytoplasmic membranes. Collectively, these results indicate that naegleriapore isoforms with antibacterial and pore-forming activity are proteolytically released from the same precursor protein, presumably to generate a phylogenetically ancient complementary antimicrobial arsenal.


Asunto(s)
Antiinfecciosos/farmacología , Naegleria fowleri/metabolismo , Péptidos/química , Péptidos/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Cisteína/química , Citoplasma/metabolismo , Relación Dosis-Respuesta a Droga , Datos de Secuencia Molecular , Isoformas de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo
9.
J Biol Chem ; 277(36): 32596-605, 2002 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-12070154

RESUMEN

In Dictyostelium discoideum counting factor (CF), a secreted approximately 450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC(50) of approximately 3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. (125)I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have approximately 53 cell-surface sites that bind countin with a K(D) of approximately 1.5 ng/ml or 60 pm. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.


Asunto(s)
Dictyostelium/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Actinas/metabolismo , Animales , Sitios de Unión , Adhesión Celular , Movimiento Celular , Cromatografía en Gel , Clonación Molecular , AMP Cíclico/metabolismo , Citoesqueleto/metabolismo , ADN Complementario/metabolismo , Dimerización , Relación Dosis-Respuesta a Droga , Yodo/farmacología , Cinética , Miosinas/metabolismo , Fosforilación , Unión Proteica , Proteínas Recombinantes/metabolismo , Transducción de Señal , Factores de Tiempo , Ultracentrifugación
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