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1.
Org Biomol Chem ; 18(25): 4717-4722, 2020 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-32525502

RESUMEN

Methods for chemical modification of native proteins in a controlled fashion are in high demand. Here, a novel protocol that exploits bifunctional reagents for transient targeting of solvent exposed disulphides to direct the introduction of a single exogenous reactive thiol handle at a lysine side chain has been developed. The protocol has successfully been applied to functionalize six different Fabs and human growth hormone.


Asunto(s)
Disulfuros/química , Hormona del Crecimiento/química , Humanos , Lisina/química , Estructura Molecular
2.
Bioconjug Chem ; 29(9): 3129-3143, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30168709

RESUMEN

The present work describes a series of human growth hormone (hGH) albumin binder conjugates with an extended in vivo half-life. A broad range of different conjugates were studied by varying the albumin binder structure and conjugation site. Conjugates were conveniently obtained by reductive alkylation or by alkylation to introduced cysteines using functionalized albumin-binding side chains. In vitro and in vivo profiling provided the basis for identification of position L101C in human growth hormone as the most optimal position for conjugation, where both a sufficient level of receptor binding and a suitably long half-life could yield a molecule with potential for a once-weekly dosing regimen.


Asunto(s)
Albúminas/metabolismo , Hormona de Crecimiento Humana/metabolismo , Alquilación , Animales , Área Bajo la Curva , Semivida , Oxidación-Reducción , Unión Proteica , Ratas , Ratas Sprague-Dawley
3.
Artículo en Inglés | MEDLINE | ID: mdl-17905622

RESUMEN

An extract of the skin of the Japanese tree frog, Hyla japonica Günther, 1859 (Anura: Hylidae) did not inhibit the growth of the bacteria Escherichia coli or Staphylococcus aureus, but contained a protein that was strongly hemolytic against human erythrocytes. The protein was purified to near homogeneity by reverse-phase HPLC, and its N-terminal amino acid sequence (SGRGKGGKGL...) identified it as histone H4. The complete primary structure of the 102-amino-acid-residue histone H4 was determined by a combination of molecular cloning of genomic and complementary DNAs encoding the protein. The molecular mass of the purified histone H4 determined by electrospray mass spectrometry was 71+/-2 Daltons greater than that predicted from the deduced amino acid sequence of the protein. The +71 mass units is consistent with the proposal that the protein isolated from the skin was post-translationally modified by addition of one acetyl and two methyl groups. The stem-loop structure at the 3' flanking region of the H. japonica histone H4 gene, which acts as a transcription termination signal, contained a nucleotide sequence (5'-GGCTCTCCTCAGAGCC-3') with unusual structural features not seen in other histone genes.


Asunto(s)
Proteínas Anfibias/aislamiento & purificación , Hemolíticos/aislamiento & purificación , Histonas/aislamiento & purificación , Piel/química , Secuencia de Aminoácidos , Proteínas Anfibias/genética , Proteínas Anfibias/farmacología , Animales , Anuros , Mezclas Complejas/química , Eritrocitos/química , Escherichia coli/crecimiento & desarrollo , Hemólisis/efectos de los fármacos , Hemolíticos/química , Hemolíticos/farmacología , Histonas/química , Histonas/genética , Histonas/farmacología , Humanos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Piel/microbiología , Staphylococcus aureus/crecimiento & desarrollo , Transcripción Genética/fisiología
4.
Peptides ; 28(6): 1268-74, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17451843

RESUMEN

The Cascades frog Rana cascadae belongs to the Amerana (or Rana boylii) group that includes six additional species from western North America (R. aurora, R. boylii, R. draytonii, R. luteiventris, R. muscosa, and R. pretiosa). R. cascadae is particularly susceptible to pathogenic microorganisms in the environment and populations have declined precipitously in parts of its range so that the protection afforded by dermal antimicrobial peptides may be crucial to survival of the species. Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of six peptides with differential cytolytic activities that were present in high abundance. Structural characterization showed that they belonged to the ranatuerin-2 (one peptide), brevinin-1 (one peptide), and temporin (four peptides) families. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAGKLLETLKCKITGC) and brevinin-1CSa (FLPILAGLAAKIVPKLFCLATKKC) showed broad spectrum antibacterial activity (MIC

Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Evolución Molecular , Ranidae/metabolismo , Secuencia de Aminoácidos , Animales , Antiinfecciosos/metabolismo , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Secuencia Conservada , Disulfuros/química , Eritrocitos/efectos de los fármacos , Humanos , Dosificación Letal Mediana , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Ranidae/clasificación , Ranidae/genética , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
5.
Biochem J ; 396(2): 391-9, 2006 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-16475979

RESUMEN

Dipeptidyl peptidases 8 and 9 have been identified as gene members of the S9b family of dipeptidyl peptidases. In the present paper, we report the characterization of recombinant dipeptidyl peptidases 8 and 9 using the baculovirus expression system. We have found that only the full-length variants of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave the peptide hormones glucagon-like peptide-1, glucagon-like peptide-2, neuropeptide Y and peptide YY with marked kinetic differences compared with dipeptidyl peptidase IV. Inhibition of dipeptidyl peptidases IV, 8 and 9 using the well-known dipeptidyl peptidase IV inhibitor valine pyrrolidide resulted in similar K(i) values, indicating that this inhibitor is non-selective for any of the three dipeptidyl peptidases.


Asunto(s)
Dipeptidasas/química , Dipeptidil Peptidasa 4/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Secuencia de Aminoácidos , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografía en Gel , Dipeptidasas/genética , Dipeptidasas/aislamiento & purificación , Dipeptidasas/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/aislamiento & purificación , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Cinética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Estructura Cuaternaria de Proteína , Pirroles/metabolismo , Pirroles/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Valina/metabolismo , Valina/farmacología
6.
Dev Comp Immunol ; 30(9): 831-42, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16330099

RESUMEN

The mountain yellow-legged frog (Rana muscosa) inhabits high elevation lakes in California that are largely undisturbed by human activities. In spite of this habitation in remote sites, populations continue to decline. Although predation by non-native fish is one cause for declines, some isolated populations in fishless lakes are suffering new declines. One possible cause of the current wave of declines is the introduction of the pathogenic chytrid fungus (Batrachochytrium dendrobatidis) which invades the adult skin to cause chytridiomycosis. In many amphibian species, the skin is protected by antimicrobial peptides secreted into the mucous. Here we show that R. muscosa produces three previously unknown antimicrobial peptides belonging to the ranatuerin-2 and temporin-1 families of antimicrobial peptides. These three peptides, along with bradykinin, are the most abundant peptides in the skin secretions detected by mass spectrometry. Natural mixtures of peptides and individual purified peptides strongly inhibit chytrid growth. The concentration of total peptides recovered from the skin of frogs following a mild norepinephrine induction is sufficient to inhibit chytrid growth in vitro. A comparison of the species susceptibility to chytridiomycosis and the antichytrid activity of peptides between R. muscosa and R. pipiens suggest that although R. muscosa produces more total skin peptides, it appears to be more vulnerable to B. dendrobatidis in nature. Possible differences in the antimicrobial peptide repertoires and life history traits of the two species that may account for differences in susceptibility are discussed.


Asunto(s)
Proteínas Anfibias/inmunología , Dermatomicosis/veterinaria , Péptidos/inmunología , Proteínas/inmunología , Ranidae/inmunología , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/aislamiento & purificación , Proteínas Anfibias/farmacología , Animales , Péptidos Catiónicos Antimicrobianos , Quitridiomicetos/crecimiento & desarrollo , Dermatomicosis/inmunología , Dermatomicosis/microbiología , Femenino , Masculino , Datos de Secuencia Molecular , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/farmacología , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/farmacología , Ranidae/microbiología , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria
7.
Protein Pept Lett ; 13(4): 411-5, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16712520

RESUMEN

Norepinephrine-stimulated skin secretions from the Sante Fe frog Leptodactylus laticeps contained high concentrations of a peptide, termed laticeptin, with the primary structure Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Leu-Ala-Gly-His-Leu-Ala-Thr-Lys-Val-Met-Asn-Lys-Leu.NH(2). Laticeptin inhibited the growth of selected Gram-negative bacteria but the lack of activity against Gram-positive bacteria and the very low hemolytic activity is probably a consequence of the weak amphipathicity of the peptide in its alpha-helical conformation.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Anuros , Eritrocitos/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Hemólisis , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular
8.
J Am Soc Mass Spectrom ; 16(4): 548-52, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15792724

RESUMEN

An N-acylated glucagon-like peptide 1 derivative was characterized by Fourier transform ion cyclotron resonance mass spectrometry. Both electron capture dissociation (ECD) and sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) were employed. While ECD revealed full sequence coverage, site of modification, branching point, structure of the palmitoylated modification, SORI-CAD produced less complete and more ambiguous information attributable to facile losses of the fatty acid group from both parent and fragments. Thus, ECD showed a superior characterization performance over SORI-CAD in analysis of N-acylated polypeptides.


Asunto(s)
Glucagón/química , Hipoglucemiantes/química , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Acilación , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón , Hipoglucemiantes/uso terapéutico
9.
Peptides ; 26(4): 597-601, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15752573

RESUMEN

During the breeding season of the mountain chicken frog Leptodactylus fallax, fighting between males results in the emergence of dominant animals that subsequently attract females to nesting sites. A peptide, termed Leptodactylus aggression-stimulating peptide (LASP), was isolated from norepinephrine-stimulated skin secretions from male specimens of L. fallax that was not present in skin secretions obtained from females. The primary structure of the peptide was established as: Gly-Leu-Trp-Asp-Asp-Leu-Lys-Ala-Ala-Ala-Lys-Lys-Val-Val-Ser-Ser-Leu-Ala-Ser-Ala-Ala-Ile-Glu-Lys-Leu NH2. LASP had no pheromone-like action on females but had a chemoattractive effect on males and stimulated aggressive behaviors, such as rearing and leaping. It is suggested that this peptide may play an important role in initiating the competitive male-male interactions that are associated with the onset of reproductive behavior in L. fallax.


Asunto(s)
Agresión , Péptidos/metabolismo , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Anuros , Cromatografía Líquida de Alta Presión , Femenino , Masculino , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/fisiología , Conducta Sexual Animal
10.
Regul Pept ; 124(1-3): 173-8, 2005 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-15544856

RESUMEN

A 25 amino-acid-residue, C-terminally alpha-amidated peptide with antimicrobial activity, which has been termed fallaxin, was isolated in high yield from the norepinephrine-stimulated skin secretions of the mountain chicken frog Leptodactylus fallax (Anura:Leptodactylidae). The amino acid sequence of the peptide (Gly-Val-Val-Asp-Ile-Leu-Lys-Gly-Ala-Ala-Lys-Asp-Ile-Ala-Gly-His-Leu-Ala-Ser-Lys-Val-Met-Asn-Lys-Leu.NH2) shows structural similarity with members of the ranatuerin-2 family previously isolated from the skins of frogs of the genus Rana that are only distantly related to the Leptodactylidae. This observation is consistent with the hypothesis that many frog skin antimicrobial peptides are related evolutionarily, having arisen from multiple duplications of an ancestral gene that existed before the radiation of the different families. Fallaxin inhibited the growth of reference strains of Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) but with relatively low potency (MIC> or =20 microM) and was inactive against the Gram-positive bacterium (Staphylococcus aureus) and the yeast Candida albicans. The hemolytic activity of fallaxin was very low (HC50>200 microM). A second peptide, comprising residues (1-22) of fallaxin, was also isolated from the skin secretions but this component was inactive against the microorganisms tested.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , Anuros/metabolismo , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Eritrocitos/efectos de los fármacos , Femenino , Hemólisis/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Piel/química
11.
FEBS Lett ; 555(3): 478-82, 2003 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-14675759

RESUMEN

A commercial antibody (clone 22) directed against the apoptosis-linked gene 2 (alg2, pdcd6) encoded protein has been used by several groups. Up-regulated expression of the antigen was observed in primary tumours and in metastatic tissue and also during rat brain ischemia. Furthermore, antigen down-regulation was found in human atherosclerotic plaques. Recently, we found that the clone 22 antibody does not recognise ALG-2. In the present study the antigen of the clone 22 antibody was identified as the heat shock protein 90 (HSP90) co-chaperone protein p23, identical to the cytosolic prostaglandin E2 synthase, by immunoprecipitation followed by tryptic in-gel digests and mass spectrometry of the purified peptides. Moreover, the heterogeneous ribonuclear protein A2/B1 was found to be a part of the p23 co-immunoprecipitated protein complex.


Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Animales , Anticuerpos/inmunología , Apoptosis , Proteínas Reguladoras de la Apoptosis , Proteínas de Unión al Calcio/inmunología , Clonación Molecular , Regulación de la Expresión Génica , Humanos , Oxidorreductasas Intramoleculares , Células Jurkat , Ratones , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/genética , Pruebas de Precipitina , Prostaglandina-E Sintasas , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Receptor fas/inmunología
12.
Regul Pept ; 118(3): 135-41, 2004 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15003829

RESUMEN

Nine peptides displaying varying degrees of antimicrobial activity were extracted from the skin of the Hokkaido frog, Rana pirica. Five structurally related peptides were identified as members of the brevinin-2 family. These peptides were active against reference strains of Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae) and Gram-positive (Staphlococcus aureus) bacteria but displayed relatively low hemolytic activity. The most abundant peptide, brevinin-2PRa (680 nmol/g weight of dry skin) showed high potency [minimal inhibitory concentration (MIC) values between 6 and 12 microM] against a range of clinical isolates of P. aeruginosa. In addition, activity was unaffected by NaCl concentrations up to 200 mM. Cladistic analysis based on the primary structures of brevinin-2 peptides supports a close phylogenetic relationship between R. pirica and Japanese mountain brown frog Rana ornativentris. One peptide of the ranatuerin-2 family and one strongly hemolytic peptide of the brevinin-1 family were also isolated from the extract along with two members of the temporin family, temporin-1PRa (ILPILGNLLNGLL.NH(2)) and temporin-1PRb (ILPILGNLLNSLL.NH(2)) that atypically lacked basic amino acid residues and showed only very weak antimicrobial and hemolytic activity.


Asunto(s)
Proteínas Anfibias/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Piel/microbiología , Secuencia de Aminoácidos , Proteínas Anfibias/genética , Proteínas Anfibias/aislamiento & purificación , Animales , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Candida albicans/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Farmacorresistencia Bacteriana , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Péptidos , Filogenia , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/farmacología , Ranidae , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
13.
Artículo en Inglés | MEDLINE | ID: mdl-24463457

RESUMEN

The primary structures of host-defense peptides present in frog skin secretions constitute useful molecular markers for establishing taxonomic classifications and investigating phylogenetic relationships between species within a particular genus. Peptidomic analysis has led to the characterization of multiple host-defense peptides in norepinephrine-stimulated skin secretions of three species of frogs from the family Ranidae: Lithobates forreri (Boulenger, 1883), Hylarana luctuosa (Peters, 1871), and Hylarana signata (Günther, 1872). The L. forreri secretions contain ranatuerin-2 (2 peptides), brevinin-1 (4 peptides), and temporin (1 peptide). The H. luctuosa secretions contain brevinin-2 (4 peptides), esculentin-1 (1 peptide), esculentin-2 (1 peptide), palustrin-2 (2 peptides), and temporin (2 peptides). The H. signata secretions contain brevinin-2 (4 peptides), brevinin-1 (5 peptides), palustrin-2 (1 peptide), and temporin (2 peptides). Cladistic analysis based upon the primary structures of 44 ranatuerin-2 peptides from 20 Lithobates species indicates a close phylogenetic relationship between L. forreri, Lithobates onca, and Lithobates yavapaiensis. A similar cladistic analysis based upon the primary structures of 27 brevinin-2 peptides from 8 Hylarana species provides support for a close phylogenetic relationship between H. signata and Hylarana picturata, while showing that the species are not conspecific, with H. luctuosa more distantly related.


Asunto(s)
Proteínas Anfibias/química , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas/química , Ranidae/genética , Secuencia de Aminoácidos , Proteínas Anfibias/genética , Proteínas Anfibias/aislamiento & purificación , Proteínas Anfibias/farmacología , Animales , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/tratamiento farmacológico , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Filogenia , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas/farmacología , Piel/química
14.
Peptides ; 40: 65-71, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23262358

RESUMEN

Norepinephrine-stimulated skin secretions of the Tyrrhenian painted frog Discoglossus sardus Tschudi, 1837 (Alytidae) did not contain any peptide with antimicrobial or hemolytic activity. However, peptidomic analysis of the secretions revealed the presence of an abundant peptide with structural similarity to frenatin 2, previously isolated from the Australian frog Litoria infrafrenata (Hylidae). The primary structure of the peptide, termed frenatin 2D, was established as DLLGTLGNLPLPFI.NH2 by automated Edman degradation and mass spectrometry with electron-transfer dissociation (ETD)-based fragmentation and confirmed by chemical synthesis. The structure of a second frenatin 2-related peptide, termed frenatin 2.1D, that was present in much lower abundance was established as GTLGNLPAPFPG. Frenatin 2D (20 µg/ml) significantly stimulated production of the proinflammatory cytokines TNF-α (P<0.05) and IL-1ß (P<0.01) by mouse peritoneal macrophages but the peptide did not potentiate the stimulation produced by lipopolysaccharide (LPS). The peptide increased IL-12 production in both unstimulated (P<0.01) and LPS-stimulated (P<0.05) cells but stimulatory effects on IL-6 production were not significant. The biological role of frenatin 2D is unknown but it is speculated that the peptide acts on skin macrophages to produce a cytokine-mediated stimulation of the adaptive immune system in response to invasion by microorganisms.


Asunto(s)
Proteínas Anfibias/metabolismo , Interleucina-1beta/biosíntesis , Péptidos/administración & dosificación , Péptidos/aislamiento & purificación , Piel , Factor de Necrosis Tumoral alfa/biosíntesis , Inmunidad Adaptativa , Animales , Antiinfecciosos/administración & dosificación , Antiinfecciosos/química , Antiinfecciosos/aislamiento & purificación , Anuros , Australia , Bacterias/efectos de los fármacos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Espectrometría de Masas , Ratones , Péptidos/química , Piel/química , Piel/metabolismo
15.
Peptides ; 31(6): 989-94, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20226221

RESUMEN

The Volcano clawed frog Xenopus amieti Kobel, du Pasquier, Fischberg, and Gloor, 1980, with a chromosome number of 2n=72, is believed to have undergone two rounds of genome duplication since evolving from a diploid ancestor. Nine peptides with differential antimicrobial activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of X. amieti that showed structural similarity to peptides previously isolated from the tetraploid frog X. laevis (2n=36) and the diploid frog Silurana (formerly Xenopus) tropicalis (2n=20). Two peptides (magainin-AM1 and -AM2) are othologous to the magainins, two peptides (PGLa-AM1 and -AM2) orthologous to peptide glycine-leucine-amide, four peptides (CPF-AM1, -AM2, -AM3, -AM4) orthologous to caerulein-precursor fragments, and one peptide (XPF-AM1) structurally similar to xenopsin-precursor fragments were characterized. CFP-AM1 (GLGSVLGKALKIGANLL.NH(2)) was the most potent peptide present in the secretions and magainin-AM2 (GVSKILHSAGKFGKAFLGEIMKS) was the most abundant. The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage. However, the very low antimicrobial activity of the magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).


Asunto(s)
Magaininas/aislamiento & purificación , Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Antiinfecciosos/análisis , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Escherichia coli/efectos de los fármacos , Magaininas/química , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Norepinefrina , Péptidos/análisis , Péptidos/química , Péptidos/genética , Piel/química , Piel/efectos de los fármacos , Piel/metabolismo , Staphylococcus aureus/efectos de los fármacos , Xenopus
16.
Peptides ; 30(6): 1069-73, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19463738

RESUMEN

Two families of structurally related C-terminally alpha-amidated antimicrobial peptides have been identified in norepinephrine-stimulated skin secretions of the midwife toad Alytes obstetricans (Alytidae). The alyteserin-1 peptides (Gly-Leu-Lys-(Asp/Glu)-Ile-Phe-Lys-Ala-Gly-Leu-Gly-Ser-Leu-Val-Lys-(Gly/Asn)-Ile-Ala-Ala-His-Val-Ala-(Asn/Ser).NH(2)) show limited structural similarity to the ascaphins from the skins of frogs of the family Leiopelmatidae. Alyteserin-2a (Ile-Leu-Gly-Lys-Leu-Leu-Ser-Thr-Ala-Ala-Gly-Leu-Leu-Ser-Asn-Leu.NH(2)) and alyteserin-2b and -2c (Ile-Leu-Gly-Ala-Ile-Leu-Pro-Leu-Val-Ser-Gly-Leu-Leu-Ser-(Asn/Ser)-Lys-Leu x NH(2)) show limited sequence identity with bombinin H6, present in the skins of frogs of the family Bombinatoridae. The alyteserin-1 peptides show selective growth inhibitory activity against the Gram-negative bacteria Escherichia coli (MIC=25 microM) whereas alyteserin-2a is more potent against the Gram-positive bacteria Staphylococcus aureus (MIC=50 microM). The hemolytic activity against human erythrocytes of all peptides tested is relatively weak (LC(50)>100 microM). The data demonstrate that the frogs belonging to the family Alytidae are among those producing dermal antimicrobial peptides that may represent a component of the animal's system of innate immunity.


Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Anuros , Eritrocitos/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Piel/metabolismo , Staphylococcus aureus/efectos de los fármacos , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Humanos , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Piel/química , Factores de Tiempo
17.
Artículo en Inglés | MEDLINE | ID: mdl-16928470

RESUMEN

Four structurally-related peptides with antimicrobial activity were isolated from an extract of the skin of the Chinese brown frog, Rana chaochiaoensis Liu, 1946. Determination of their primary structures revealed that they are members of the japonicin-2 family, previously identified only in the skin of the Japanese brown frog, R. japonica. Japonicin-2CHa (FVLPLLGILPKELCIVLKKNC) represented the most abundant peptide in the extract but its growth-inhibitory potency against Staphylococcus aureus (minimum inhibitory concentration, MIC=100 microM) and Escherichia coli (MIC>100 microM) was appreciably less than that of the more cationic japonicin-2 (FGLPMLSILPKALCILLKRKC). The high degree of structural similarity of japonicin-2CHb (VVPAFVLLKKAICIMLKRNC) with japonicin-2CHc (K9 --> R), and japonicin-2CHd (L16 --> F) is suggestive of recent gene duplication events. The data indicate a close phylogenetic relationship between R. chaochiaoensis and R. japonica but demonstrate that the species are not conspecific.


Asunto(s)
Proteínas Anfibias/aislamiento & purificación , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Ranidae , Piel/metabolismo , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Escherichia coli/efectos de los fármacos , Femenino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptidos , Homología de Secuencia de Aminoácido , Staphylococcus aureus/efectos de los fármacos
18.
Artículo en Inglés | MEDLINE | ID: mdl-16236555

RESUMEN

Norepinephrine-stimulated skin secretions were obtained from male specimens of the South American bullfrog, Leptodactylus pentadactylus and shown to contain two peptides that inhibited the growth of microorganisms. The primary structure of a previously undescribed peptide, termed pentadactylin, was established as Gly-Leu-Leu-Asp-Thr-Leu-Lys-Gly-Ala-Ala-Lys-Asn-Val-Val-Gly-Ser-Leu-Ala-Ser-Lys-Val-Met-Glu-Lys-Leu.NH2. The second peptide, which differs from pentadactylin by eight amino acid residues, is identical to fallaxin previously isolated from skin secretions of the Caribbean mountain chicken frog L. fallax. Pentadactylin inhibited the growth of reference strains of both Gram-negative bacteria (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus group B) but potencies were relatively low (MIC values in the range 25-200 microM). The peptide showed very low hemolytic activity against human erythrocytes (LD50>400 microM).


Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Rana catesbeiana/metabolismo , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Hemólisis/efectos de los fármacos , Masculino , Datos de Secuencia Molecular
19.
Biochemistry ; 43(44): 14096-103, 2004 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-15518559

RESUMEN

Coagulation factor VIIa (FVIIa) belongs to the chymotrypsin family of S1 peptidases, whose members require the cleavage of at least one internal peptide bond to attain an active conformation. FVIIa also requires association with tissue factor (TF) to attain full catalytic competency. Without this, FVIIa has very low activity toward peptide and physiologic substrates. Reregistration of beta strands has been suggested to play a part in the activation of FVII, and their positioning is possibly important for the active conformation of FVIIa. To scrutinize this hypothesis, we have designed FVIIa variants which prevent beta strand movement and lock FVIIa in the alleged active conformation. The V299M mutation, alone or combined with the L280I mutation, was introduced to alter the first of two Leu-X-Val motifs in beta strand B2 and thereby prevent reregistration. Along the same line, C164V/V299C-FVIIa has a new disulfide which would keep beta strand B2 in the registration of active FVIIa. The amidolytic and proteolytic activities of V299M-, L280I/V299M-, and C164V/V299C-FVIIa were indistinguishable from or lower than those of wild-type FVIIa, and none of the mutants displayed an altered exposure of the N-terminal amino group of the protease domain. Moreover, the affinities of mutant and native FVIIa for TF increased to a similar extent upon incorporation of an active site inhibitor, and the enzymatic activities were equally stimulated by TF. In conclusion, we found no evidence that the mutants were in a more active state than native FVIIa. Thus, the proposed beta strand reregistration, if part of the regulatory mechanism governing FVIIa activity, apparently does not suffice for the transformation of FVIIa into an enzymatically active conformation. Our data raise the possibility that the structural differences between enzymatically latent (zymogen-like) and active FVIIa resemble those between trypsinogen and trypsin.


Asunto(s)
Precursores Enzimáticos/química , Factor VIIa/química , Sitio Alostérico , Secuencia de Aminoácidos , Animales , Línea Celular , Compuestos Cromogénicos/química , Cricetinae , Cisteína/genética , Activación Enzimática/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Factor VIIa/genética , Factor VIIa/aislamiento & purificación , Factor VIIa/metabolismo , Variación Genética , Humanos , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Especificidad por Sustrato/genética , Termodinámica , Tromboplastina/metabolismo , Transfección , Valina/genética
20.
Artículo en Inglés | MEDLINE | ID: mdl-15050930

RESUMEN

A single peptide with antimicrobial activity was extracted from the skin of the European agile frog (R. dalmatina). The primary structure of this 17 amino-acid-residue peptide (ILPLLLGKVVCAITKKC) does not immediately suggest membership of any of the previously described families of antimicrobial peptides from ranid frogs. However, if it is assumed that the peptide has undergone several residue deletions during the course of speciation, it shows sequence similarity with peptides belonging to the widely distributed brevinin-1 family, particularly those isolated from the related species Rana temporaria. The minimum inhibitory concentration of the peptide, termed brevinin-1 Da, against the Gram-positive bacterium Staphylococcus aureus was 7 microM and against the Gram-negative bacterium Escherichia coli was 30 microM.


Asunto(s)
Proteínas Anfibias/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Ranidae , Piel/química , Secuencia de Aminoácidos , Proteínas Anfibias/química , Proteínas Anfibias/genética , Animales , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Staphylococcus aureus/efectos de los fármacos
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