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1.
J Helminthol ; 90(3): 284-93, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-25761568

RESUMEN

Hymenolepis diminuta is a natural parasite of the common brown rat Rattus norvegicus, and provides a convenient model system for the assessment of the anthelmintic activity of novel drugs against cestodes. The experiments described in this paper indicate that treatment of rats infected with H. diminuta with a supernatant extract of papaya latex, containing a mixture of four cysteine proteinases, was moderately efficacious, resulting in a significant, but relatively small, reduction in worm burden and biomass. However, faecal egg output was not affected by treatment. In our experiments these effects were only partially dose-dependent, although specific inhibition by E-64 confirmed the role of cysteine proteinases as the active principles in papaya latex affecting worm growth but not statistically reducing worm burden. Data collected for a further 7 days after treatment indicated that the effects of papaya latex supernatant on worm loss and on worm growth were not enhanced. Our findings provide a starting point for further refinement in formulation and delivery, or assessment of alternative natural plant-derived cysteine proteinases in efforts to develop these naturally occurring enzymes into broad-spectrum anthelmintics, with efficacy against cestodes as well as nematodes.


Asunto(s)
Antihelmínticos/farmacología , Proteasas de Cisteína/farmacología , Himenolepiasis/veterinaria , Hymenolepis diminuta/efectos de los fármacos , Extractos Vegetales/farmacología , Enfermedades de los Roedores/tratamiento farmacológico , Animales , Antihelmínticos/administración & dosificación , Antihelmínticos/aislamiento & purificación , Carica/química , Proteasas de Cisteína/administración & dosificación , Proteasas de Cisteína/aislamiento & purificación , Himenolepiasis/tratamiento farmacológico , Masculino , Recuento de Huevos de Parásitos , Carga de Parásitos , Extractos Vegetales/administración & dosificación , Extractos Vegetales/aislamiento & purificación , Proteínas de Plantas/administración & dosificación , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/farmacología , Ratas , Resultado del Tratamiento
2.
J Helminthol ; 89(2): 165-74, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24176056

RESUMEN

We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections.


Asunto(s)
Actinidia/química , Ananas/química , Antihelmínticos/farmacología , Carica/química , Proteasas de Cisteína/farmacología , Intestinos/parasitología , Extractos Vegetales/farmacología , Estrongiloidiasis/parasitología , Animales , Antihelmínticos/aislamiento & purificación , Proteasas de Cisteína/aislamiento & purificación , Femenino , Frutas/química , Humanos , Masculino , Ratones Endogámicos C3H , Extractos Vegetales/aislamiento & purificación , Strongyloides/efectos de los fármacos
3.
Vet Parasitol ; 201(1-2): 48-58, 2014 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-24462509

RESUMEN

Little is known about the efficacy of cysteine proteinases (CP) as anthelmintics for cestode infections. We examined the effects of CPs on two rodent cestodes, Hymenolepis diminuta and H. microstoma in vitro. Our data showed that naturally occurring mixtures of CPs, such as those found in papaya latex, and relatively pure preparations of fruit bromelain, papain and stem bromelain, were active in vitro against both juvenile, artificially excysted scoleces, as well as against adult worms of both rodent cestodes. Significant dose-dependent reduction in motility, ultimately leading to death of the worms, was observed with both species, and against both freshly excysted scoleces and 14-day old pre-adult worms. The most effective was fruit bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=63 and 74 µM, respectively, and for pre-adult worms=199 and 260 µM, respectively). The least effective was stem bromelain (after 30 min of incubation of juvenile H. diminuta and H. microstoma IC50=2855 and 2772 µM, respectively, and for pre-adult worms=1374 and 1332 µM, respectively) and the efficacies of papaya latex supernatant and papain were between these extremes. In all cases these values are higher than those reported previously for efficacy of CPs against intestinal nematodes, and in contrast to nematodes, all CPs were effective against cestodes in the absence of exogenous cysteine in incubation media. The CPs appeared to attack the tegument resulting in generalised erosion mainly on the strobila. The scolex was more resistant to CP attack but nevertheless some damage to the tegument on the scolex was detected.


Asunto(s)
Antihelmínticos/farmacología , Proteasas de Cisteína/farmacología , Hymenolepis diminuta/efectos de los fármacos , Hymenolepis/efectos de los fármacos , Animales , Bromelaínas/farmacología , Carica/química , Concentración 50 Inhibidora , Estadios del Ciclo de Vida/efectos de los fármacos , Microscopía Electrónica de Rastreo , Actividad Motora/efectos de los fármacos , Papaína/farmacología
4.
J Helminthol ; 86(3): 311-6, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21794201

RESUMEN

In earlier studies of the anthelmintic activity of plant cysteine proteinases (CPs), a period of food deprivation was routinely employed before administration of CPs, but there has been no systematic evaluation as to whether this does actually benefit the anthelmintic efficacy. Therefore, we assessed the effect of fasting on the efficacy of CPs from papaya latex (PL) against Heligmosomoides bakeri in C3H mice. We used a refined, supernatant extract of papaya latex (PLS) with known active enzyme content. The animals were divided into three groups (fasted prior to treatment with PLS, not fasted but treated with PLS and fasted but given only water). The study demonstrated clearly that although food deprivation had been routinely employed in much of the earlier work on CPs in mice infected with nematodes, fasting has no beneficial effect on the efficacy of PLS against H. bakeri infections. Administration of CPs to fed animals will also reduce the stress associated with fasting.


Asunto(s)
Carica/enzimología , Proteasas de Cisteína/farmacología , Ayuno/metabolismo , Heligmosomatoidea/crecimiento & desarrollo , Extractos Vegetales/farmacología , Infecciones por Strongylida/tratamiento farmacológico , Animales , Heces/parasitología , Masculino , Ratones , Ratones Endogámicos C3H , Recuento de Huevos de Parásitos , Infecciones por Strongylida/metabolismo
5.
Parasitology ; 134(Pt 12): 1831-8, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17640402

RESUMEN

Cysteine proteinases from the fruit and latex of plants, such as papaya, pineapple and fig, have previously been shown to have substantial anthelmintic efficacy, in vitro and in vivo, against a range of animal parasitic nematodes. In this paper, we describe the in vitro effects of these plant extracts against 2 sedentary plant parasitic nematodes of the genera Meloidogyne and Globodera. All the plant extracts examined caused digestion of the cuticle and decreased the activity of the tested nematodes. The specific inhibitor of cysteine proteinases, E-64, blocked this activity completely, indicating that it was essentially mediated by cysteine proteinases. In vitro, plant cysteine proteinases are active against second-stage juveniles of M. incognita and M. javanica, and some cysteine proteinases also affect the second-stage juveniles of Globodera rostochiensis. It is not known yet whether these plant extracts will interfere with, or prevent invasion of, host plants.


Asunto(s)
Antinematodos/farmacología , Cisteína Endopeptidasas/farmacología , Magnoliopsida/química , Magnoliopsida/enzimología , Extractos Vegetales/farmacología , Tylenchoidea/efectos de los fármacos , Actinidia/química , Actinidia/enzimología , Ananas/química , Ananas/enzimología , Animales , Carica/química , Carica/enzimología , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Leucina/análogos & derivados , Leucina/farmacología , Magnoliopsida/parasitología , Microscopía Electrónica de Rastreo , Factores de Tiempo
6.
Parasitology ; 134(Pt 10): 1409-19, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17475089

RESUMEN

Gastrointestinal (GI) nematodes are important disease-causing organisms, controlled primarily through treatment with synthetic drugs, but the efficacy of these drugs has declined due to widespread resistance, and hence new drugs, with different modes of action, are required. Some medicinal plants, used traditionally for the treatment of worm infections, contain cysteine proteinases known to damage worms irreversibly in vitro. Here we (i) confirm that papaya latex has marked efficacy in vivo against the rodent gastrointestinal nematode, Heligmosomoides polygyrus, (ii) demonstrate the dose-dependent nature of the activity (>90% reduction in egg output and 80% reduction in worm burden at the highest active enzyme concentration of 133 nmol), (iii) establish unequivocally that it is the cysteine proteinases that are the active principles in vivo (complete inhibition of enzyme activity when pre-incubated with the cysteine proteinase-specific inhibitor, E-64) and (iv) show that activity is confined to worms that are in the intestinal lumen. The mechanism of action was distinct from all current synthetic anthelmintics, and was the same as that in vitro, with the enzymes attacking and digesting the protective cuticle. Treatment had no detectable side-effects on immune cell numbers in the mucosa (there was no difference in the numbers of mast cells and goblet cells between the treated groups) and mucosal architecture (length of intestinal villi). Only the infected and untreated mice had much shorter villi than the other 3 groups, which was a consequence of infection and not treatment. Plant-derived cysteine proteinases are therefore prime candidates for development as novel drugs for the treatment of GI nematode infections.


Asunto(s)
Antihelmínticos/farmacología , Carica/química , Cisteína Endopeptidasas/farmacología , Mucosa Intestinal/parasitología , Nematospiroides dubius/efectos de los fármacos , Extractos Vegetales/farmacología , Infecciones por Strongylida/tratamiento farmacológico , Animales , Carica/enzimología , Cisteína Endopeptidasas/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Células Caliciformes/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Larva/efectos de los fármacos , Masculino , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Factores Sexuales , Agua/farmacología
7.
Parasitology ; 134(Pt 1): 103-12, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17032468

RESUMEN

Cysteine proteinases from the fruit and latex of plants, including papaya, pineapple and fig, were previously shown to have a rapid detrimental effect, in vitro, against the rodent gastrointestinal nematodes, Heligmosomoides polygyrus (which is found in the anterior small intestine) and Trichuris muris (which resides in the caecum). Proteinases in the crude latex of papaya also showed anthelmintic efficacy against both nematodes in vivo. In this paper, we describe the in vitro and in vivo effects of these plant extracts against the rodent nematode, Protospirura muricola, which is found in the stomach. As in earlier work, all the plant cysteine proteinases examined, with the exception of actinidain from the juice of kiwi fruit, caused rapid loss of motility and digestion of the cuticle, leading to death of the nematode in vitro. In vivo, in contrast to the efficacy against H. polygyrus and T. muris, papaya latex only showed efficacy against P. muricola adult female worms when the stomach acidity had been neutralized prior to administration of papaya latex. Therefore, collectively, our studies have demonstrated that, with the appropriate formulation, plant cysteine proteinases have efficacy against nematodes residing throughout the rodent gastrointestinal tract.


Asunto(s)
Antinematodos/farmacología , Cisteína Endopeptidasas/farmacología , Frutas/enzimología , Infecciones por Spirurida/tratamiento farmacológico , Spiruroidea/efectos de los fármacos , Estómago/parasitología , Animales , Cisteína Endopeptidasas/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Movimiento/efectos de los fármacos , Papaína/farmacología , Infecciones por Spirurida/parasitología , Spiruroidea/fisiología
8.
Parasitology ; 132(Pt 5): 681-9, 2006 May.
Artículo en Inglés | MEDLINE | ID: mdl-16448585

RESUMEN

Extracts of plants, such as papaya, pineapple and fig, are known to be effective at killing intestinal nematodes that inhabit anterior sites in the small intestine, such as Heligmosomoides polygyrus. In this paper, we demonstrate that similar in vitro efficacy also occurs against a rodent nematode of the large intestine, Trichuris muris, and confirm that the cysteine proteinases present in the plant extracts are the active principles. The mechanism of action of these enzymes involved an attack on the structural proteins of the nematode cuticle, which was similar to that observed with H. polygyrus. However, not all plant cysteine proteinases were equally efficacious because actinidain, from the juice of kiwi fruit, had no detrimental effect on either the motility of the worms or the nematode cuticle. Papaya latex was also shown to significantly reduce both worm burden and egg output of mice infected with adult T. muris, demonstrating that enzyme activity survived passage to the caecum and was not completely inactivated by the acidity of the host's stomach or destroyed by the gastric or pancreatic proteinases. Thus, the cysteine proteinases from plants may be a much-needed alternative to currently available anthelmintic drugs due to their efficacy and novel mode of action against different gastrointestinal nematode species.


Asunto(s)
Antihelmínticos/farmacología , Cisteína Endopeptidasas/farmacología , Parasitosis Intestinales/parasitología , Fitoterapia , Tricuriasis/parasitología , Trichuris/efectos de los fármacos , Actinidia/química , Actinidia/enzimología , Ananas/química , Ananas/enzimología , Animales , Antihelmínticos/uso terapéutico , Carica/química , Carica/enzimología , Cisteína Endopeptidasas/aislamiento & purificación , Cisteína Endopeptidasas/uso terapéutico , Femenino , Ficus/química , Ficus/enzimología , Parasitosis Intestinales/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Movimiento/efectos de los fármacos , Recuento de Huevos de Parásitos , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Tricuriasis/tratamiento farmacológico , Trichuris/ultraestructura
9.
Parasitology ; 130(Pt 2): 203-11, 2005 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-15727070

RESUMEN

We examined the mechanism of action and compared the anthelmintic efficacy of cysteine proteinases from papaya, pineapple, fig, kiwi fruit and Egyptian milkweed in vitro using the rodent gastrointestinal nematode Heligmosomoides polygyrus. Within a 2 h incubation period, all the cysteine proteinases, with the exception of the kiwi fruit extract, caused marked damage to the cuticle of H. polygyrus adult male and female worms, reflected in the loss of surface cuticular layers. Efficacy was comparable for both sexes of worms, was dependent on the presence of cysteine and was completely inhibited by the cysteine proteinase inhibitor, E-64. LD50 values indicated that the purified proteinases were more efficacious than the proteinases in the crude latex, with purified ficin, papain, chymopapain, Egyptian milkweed latex extract and pineapple fruit extract containing fruit bromelain, having the most potent effect. The mechanism of action of these plant enzymes (i.e. an attack on the protective cuticle of the worm) suggests that resistance would be slow to develop in the field. The efficacy and mode of action make plant cysteine proteinases potential candidates for a novel class of anthelmintics urgently required for the treatment of humans and domestic livestock.


Asunto(s)
Antihelmínticos/farmacología , Cisteína Endopeptidasas/farmacología , Parasitosis Intestinales/parasitología , Leucina/análogos & derivados , Nematodos/efectos de los fármacos , Infecciones por Nematodos/parasitología , Actinidia/enzimología , Ananas/enzimología , Animales , Asclepias/enzimología , Carica/enzimología , Inhibidores de Cisteína Proteinasa/farmacología , Femenino , Ficus/enzimología , Humanos , Leucina/farmacología , Masculino , Ratones , Microscopía Electrónica de Rastreo , Nematodos/aislamiento & purificación , Nematodos/ultraestructura , Papaína/farmacología , Extractos Vegetales/antagonistas & inhibidores , Extractos Vegetales/farmacología , Proteínas de Plantas/farmacología
11.
Arch Biochem Biophys ; 303(2): 208-13, 1993 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-8512309

RESUMEN

Two endopeptidases are present in the seeds of Vigna aconitifolia (moth bean), and their activities increase during germination. One enzyme, which we term "vignain," can be assayed with benzyloxycarbonyl-phenylalanyl-arginyl-7-(4-methyl)coumarylamide as substrate. The second is legumain (EC 3.4.22.34), which can be assayed with benzyloxycarbonyl-alanyl-alanyl-asparaginyl-7-(4-methyl)-coumarylamide. The enzymes were purified, and their specificities for substrates and inhibitors were examined. Vignain has properties expected of a cysteine endopeptidase of the papain family, with the exception of a remarkably low reactivity with iodoacetate. Legumain is a very atypical cysteine endopeptidase, being insensitive to inhibition by chicken cystatin and E-64 (L-3-carboxy-2,3-trans-epoxypropionyl-leucyl-amido(4-guanidino )butane), and reacting more rapidly with iodoacetamide than with iodoacetate. We discuss our findings in relation to the literature on the proteolytic enzymes of legume seeds.


Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Fabaceae/enzimología , Proteínas de Plantas , Plantas Medicinales , Semillas/enzimología , Secuencia de Aminoácidos , Cistatinas/farmacología , Cisteína Endopeptidasas/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Yodoacetamida/farmacología , Yodoacetatos/farmacología , Ácido Yodoacético , Leucina/análogos & derivados , Leucina/farmacología , Datos de Secuencia Molecular , Peso Molecular , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Especificidad por Sustrato
12.
FEBS Lett ; 269(2): 328-30, 1990 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-2401357

RESUMEN

The inhibitory specificity of a protein from potato tubers that inhibits cysteine proteinases (potato cysteine proteinase inhibitor, PCPI) has been compared with that of chicken egg-white cystatin. Most proteinases that are inhibited by cystatin were also inhibited by PCPI, but the potato inhibitor inhibited stem bromelain and fruit bromelain, which are not inhibited by cystatin, and for which no protein inhibitor of comparable potency has previously been described. In contrast, papaya proteinase IV was unaffected by PCPI as it is by the cystatins, and the exopeptidase, dipeptidyl peptidase I, is inhibited by cystatins, but was unaffected by PCPI. The differences in inhibitory specificity between these proteins may well reflect differences between superfamilies of cysteine proteinase inhibitors.


Asunto(s)
Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Cistatinas/farmacología , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Cinética , Solanum tuberosum
13.
FEBS Lett ; 267(1): 13-5, 1990 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-2365079

RESUMEN

The amino acid sequence of a cathepsin D inhibitor isolated from potato is described. It was determined by analysis of peptides generated by use of the glycine-specific proteinase PPIV. The order of the peptides was established by examination of tryptic peptides derived from the two cyanogen bromide peptides. The inhibitor comprises 187 amino acid residues, and has a calculated Mr of 20,450.


Asunto(s)
Catepsina D , Inhibidores de Proteasas/análisis , Solanum tuberosum/enzimología , Secuencia de Aminoácidos , Catepsina D/antagonistas & inhibidores , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Datos de Secuencia Molecular
14.
Biochem J ; 266(3): 869-75, 1990 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-2327970

RESUMEN

The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.


Asunto(s)
Cisteína Endopeptidasas/análisis , Plantas/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bromelaínas/aislamiento & purificación , Pollos , Cromatografía por Intercambio Iónico , Cistatinas/farmacología , Inhibidores de Cisteína Proteinasa , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular
15.
FEBS Lett ; 247(2): 419-24, 1989 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-2714443

RESUMEN

The amino acid sequence of stem bromelain, the major cysteine proteinase from pineapple stem is described. It shows that the enzyme is a member of the papain superfamily of cysteine proteinases, but is not very closely related to any other known member of this group. The sequence shows mutation or deletion of several residues that have been conserved in cysteine proteinases examined previously, including Asn-175 (papain). We suggest that some of these changes have the effect of altering the active-site geometry of stem bromelain, and that this accounts for the resistance of the enzyme to inhibition by cystatins and E-64[L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)b utane].


Asunto(s)
Bromelaínas/metabolismo , Cistatinas , Cisteína Endopeptidasas , Endopeptidasas , Plantas/enzimología , Proteínas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Bromelaínas/antagonistas & inhibidores , Catepsina H , Catepsina L , Catepsinas , Bromuro de Cianógeno , Frutas , Humanos , Leucina/análogos & derivados , Leucina/farmacología , Datos de Secuencia Molecular , Estructura Molecular , Papaína , Fragmentos de Péptidos , Inhibidores de Proteasas , Proteínas/farmacología , Homología de Secuencia de Ácido Nucleico , Relación Estructura-Actividad
16.
Arch Biochem Biophys ; 267(1): 262-70, 1988 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-3196029

RESUMEN

A previously unknown cysteine proteinase, named ananain, has been isolated from crude commercial pineapple stem bromelain. The purification procedure involved affinity chromatography on Sepharose-Gly-Phe-glycinaldehyde semicarbazone, and cation-exchange chromatography. The relative molecular mass of ananain was very similar to that of bromelain (25,000 and 26,000, respectively), but ananain differed greatly in specificity for hydrolysis of peptide and protein substrates. The new enzyme behaved as a typical cysteine proteinase in showing strong inhibition by chicken cystatin, whereas bromelain was scarcely affected. Ananain was also shown to be immunologically distinct from bromelain. The significance of the discovery of ananain for the interpretation of previous work on "bromelain" is pointed out.


Asunto(s)
Cisteína Endopeptidasas/aislamiento & purificación , Frutas/enzimología , Sitios de Unión , Cromatografía de Afinidad , Inhibidores de Cisteína Proteinasa , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética
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