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1.
Front Chem ; 8: 624678, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33520939

RESUMEN

Arginase catalyzes the hydrolysis of l-arginine into l-ornithine and urea, acting as a key enzyme in the biosynthesis of polyamines. Leishmania growth and survival is dependent on polyamine biosynthesis; therefore, inhibition of Leishmania arginase may be a promising therapeutic strategy. Here, we evaluated a series of thirty-six chalcone derivatives as potential inhibitors of Leishmania infantum arginase (LiARG). In addition, the activity of selected inhibitors against L. infantum parasites was assessed in vitro. Seven compounds exhibited LiARG inhibition above 50% at 100 µM. Among them, compounds LC41, LC39, and LC32 displayed the greatest inhibition values (72.3 ± 0.3%, 71.9 ± 11.6%, and 69.5 ± 7.9%, respectively). Molecular docking studies predicted hydrogen bonds and hydrophobic interactions between the most active chalcones (LC32, LC39, and LC41) and specific residues from LiARG's active site, such as His140, Asn153, His155, and Ala193. Compound LC32 showed the highest activity against L. infantum promastigotes (IC50 of 74.1 ± 10.0 µM), whereas compounds LC39 and LC41 displayed the best results against intracellular amastigotes (IC50 of 55.2 ± 3.8 and 70.4 ± 9.6 µM, respectively). Moreover, compound LC39 showed more selectivity against parasites than host cells (macrophages), with a selectivity index (SI) of 107.1, even greater than that of the reference drug Fungizone®. Computational pharmacokinetic and toxicological evaluations showed high oral bioavailability and low toxicity for the most active compounds. The results presented here support the use of substituted chalcone skeletons as promising LiARG inhibitors and antileishmanial drug candidates.

2.
J Enzyme Inhib Med Chem ; 34(1): 1100-1109, 2019 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-31124384

RESUMEN

Inhibition of Leishmania arginase leads to a decrease in parasite growth and infectivity and thus represents an attractive therapeutic strategy. We evaluated the inhibitory potential of selected naturally occurring phenolic substances on Leishmania infantum arginase (ARGLi) and investigated their antileishmanial activity in vivo. ARGLi exhibited a Vmax of 0.28 ± 0.016 mM/min and a Km of 5.1 ± 1.1 mM for L-arginine. The phenylpropanoids rosmarinic acid and caffeic acid (100 µM) showed percentages of inhibition of 71.48 ± 0.85% and 56.98 ± 5.51%, respectively. Moreover, rosmarinic acid and caffeic acid displayed the greatest effects against L. infantum with IC50 values of 57.3 ± 2.65 and 60.8 ± 11 µM for promastigotes, and 7.9 ± 1.7 and 21.9 ± 5.0 µM for intracellular amastigotes, respectively. Only caffeic acid significantly increased nitric oxide production by infected macrophages. Altogether, our results broaden the current spectrum of known arginase inhibitors and revealed promising drug candidates for the therapy of visceral leishmaniasis.


Asunto(s)
Antiprotozoarios/farmacología , Arginasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Leishmania infantum/efectos de los fármacos , Fenoles/farmacología , Animales , Antiprotozoarios/química , Arginasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Leishmania infantum/enzimología , Leishmania infantum/crecimiento & desarrollo , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Fenoles/química , Células RAW 264.7 , Relación Estructura-Actividad
3.
Protein Expr Purif ; 121: 31-40, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26792557

RESUMEN

RhlR is a 241-residue quorum sensing receptor that controls the expression of a myriad of virulence genes in Pseudomonas aeruginosa. Here, the DNA sequence encoding the carboxi-terminal DNA-binding domain of RhlR was cloned into the pET-RP1B plasmid and expressed as an N-terminal fusion protein to the expression/purification Thio6His6 tag. The fusion construct expressed insolubly in Escherichia coli BL21 (DE3) cells. The recombinant protein was extracted from the bacterial inclusion bodies and refolded in the presence of the charged amino acids l-arginine and l-glutamate. The refolded protein was purified by a combination of Ni(+2)-affinity and size exclusion chromatography, allowing the production of 2 mg of highly purified protein (>95% purity) per 5 mg of wet cells derived from 1 L culture. (1)H 1D NMR analysis revealed that the recombinant protein is folded. Moreover, a fluorescence anisotropy DNA-binding assay showed that the refolded protein is functional, as it recognizes the rhlAB promoter. This is the first time that a domain of the quorum sensing regulator RhlR was produced in sufficient amounts for structural studies, enabling the investigation of the molecular basis for RhlR specific interaction with DNA promoters.


Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Percepción de Quorum/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Pliegue de Proteína , Pseudomonas aeruginosa/genética
4.
J Insect Physiol ; 59(12): 1242-9, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24140472

RESUMEN

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.


Asunto(s)
Fosfatasa Ácida/metabolismo , Yema de Huevo/metabolismo , Mariposas Nocturnas/enzimología , Animales , Desarrollo Embrionario , Polifosfatos/metabolismo , Proteolisis
5.
J Insect Physiol ; 57(7): 945-53, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21540034

RESUMEN

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host-pathogen associations and the origin of protease activity that led to yolk resorption are discussed.


Asunto(s)
Aspergillus niger/fisiología , Rhodnius/inmunología , Rhodnius/microbiología , Animales , Apoptosis , Proteasas de Ácido Aspártico/metabolismo , Proteasas de Cisteína/metabolismo , Femenino , Colorantes Fluorescentes , Atresia Folicular , Etiquetado Corte-Fin in Situ , Indoles/química , Microscopía Electrónica de Transmisión , Rhodnius/fisiología , Vitelogénesis
6.
J Insect Physiol ; 54(5): 883-91, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18499122

RESUMEN

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.


Asunto(s)
Fosfatasa Ácida/metabolismo , Cisteína Endopeptidasas/metabolismo , Periplaneta/embriología , Vitelinas/metabolismo , Factores de Edad , Animales , Cumarinas , Dipéptidos , Proteínas del Huevo/metabolismo , Ensayo de Inmunoadsorción Enzimática , Concentración de Iones de Hidrógeno , Periplaneta/metabolismo , Ácidos Fosfoaminos/metabolismo
7.
Micron ; 37(1): 41-6, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-16169237

RESUMEN

Sulfated glycosaminoglycans (GAGs) were isolated and characterized in thoracic muscle, fat body, whole digestive tract (stomach+intestine) and reproductive tract of adult male cockroaches, Periplaneta americana. Heparan sulfate (HS) was the predominant sulfated GAG species in the tissues analyzed, corresponding to more than 90% of the total sulfated GAG content. In both the thoracic muscle and fat body it was the only sulfated GAG species detected. We also determined the location of sulfated GAGs in most of these organs by histochemical analysis using 1,9-dimethylmethylene blue. In the thoracic muscle, sulfated GAG metachromatic staining was detected only in the connective tissue that surrounds the muscle bundles or fascicles. In the intestinal tract, metachromatic staining was observed in both epithelial and lining columnar cells. Only spermatozoa presented metachromatic material in the male reproductive tract. Since, HS corresponds to 90-100% of total sulfated GAGs in these tissues, the metachromatic staining specifically reflects the location of this particular sulfated GAG in these organs. In conclusion, the present study extends previous observations on the GAG composition in cockroaches providing new information on the tissue distribution and location of HS in several internal organs of adult males of the cockroach P. americana.


Asunto(s)
Glicosaminoglicanos/química , Heparitina Sulfato/metabolismo , Periplaneta/química , Animales , Sistema Digestivo/química , Sistema Digestivo/metabolismo , Cuerpo Adiposo/química , Cuerpo Adiposo/metabolismo , Genitales Masculinos/química , Genitales Masculinos/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/análisis , Heparitina Sulfato/química , Masculino , Músculos/química , Especificidad de Órganos , Especificidad de la Especie
8.
Insect Biochem Mol Biol ; 32(5): 537-45, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11891130

RESUMEN

The participation of eicosanoids and second messengers on the regulation of RHBP endocytosis by the ovaries was investigated, using [(125)I]RHBP in experiments in vivo and in vitro. Addition of PGE(2) (one of the products of the cyclooxygenase pathway) decreased in vitro the uptake of RHBP by 35%. The rate of RHBP endocytosis increased in the presence of indomethacin, a potent cyclooxigenase inhibitor, up to 50% in vitro and up to 55% in vivo, thus giving support to the role of cyclooxygenase derivatives on endocytosis regulation. The amount of PGE(2) secreted to the culture medium by the cells of Rhodnius prolixus ovaries was 1.1 ng/ovary following RHBP uptake assay. The amount of PGE(2) decreases approximately 25% in the presence of 5 microM indomethacin. Using a scanning electron microscope we have observed that neither the surface area nor the patencies of follicle cells were affected by treatment with indomethacin, thus suggesting that, its effect is elicited in the oocyte. Finally, we have identified two ovarian peptides that were dephosphorylated after the indomethacin treatment (18 and 25 kDa). Taken together these data show that local mediators such as eicosanoids act upon the oocytes controlling RHBP endocytosis, perhaps using the protein phosphorylation signal transduction pathway.


Asunto(s)
Proteínas Portadoras/metabolismo , Dinoprostona/metabolismo , Eicosanoides/metabolismo , Endocitosis/fisiología , Hemoproteínas/metabolismo , Rhodnius/metabolismo , Animales , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/antagonistas & inhibidores , Dinoprostona/farmacología , Eicosanoides/antagonistas & inhibidores , Eicosanoides/farmacología , Endocitosis/efectos de los fármacos , Femenino , Proteínas de Unión al Hemo , Indometacina/farmacología , Radioisótopos de Yodo , Técnicas de Cultivo de Órganos , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Ovario/efectos de los fármacos , Ovario/metabolismo , Fosforilación , Rhodnius/efectos de los fármacos
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