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1.
PLoS One ; 18(4): e0282277, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37098078

RESUMEN

The MRE11A-RAD50-NBS1 complex activates the ataxia-telangiectasia mutated (ATM) pathway and plays a central role in genome homeostasis. The association of RAD50 mutations with disease remains unclear; hence, we adopted a medaka rad50 mutant to demonstrate the significance of RAD50 mutation in pathogenesis using the medaka as an experimental animal. A 2-base pair deletion in the rad50 gene was introduced into transparent STIII medaka using the CRISPR/Cas9 system. The mutant was analyzed histologically for tumorigenicity and hindbrain quality, as well as for swimming behavior, to compare with existing ATM-, MRE11A-, and NBS1-mutation-related pathology. Our results revealed that the medaka rad50 mutation concurrently reproduced tumorigenesis (8 out of 10 rad50Δ2/+ medaka), had a decrease in median survival time (65.7 ± 1.1 weeks in control vs. 54.2 ± 2.6 weeks in rad50Δ2/+ medaka, p = 0.001, Welch's t-test), exhibited semi-lethality in rad50Δ2/Δ2 medaka and most of the major ataxia-telangiectasia phenotypes, including ataxia (rheotaxis ability was lower in rad50Δ2/+ medaka than in the control, Mann-Whitney U test, p < 0.05), and telangiectasia (6 out of 10 rad50Δ2/+ medaka). The fish model may aid in further understanding the tumorigenesis and phenotype of ataxia-telangiectasia-related RAD50 germline mutations and in developing novel therapeutic strategies against RAD50 molecular disorders.


Asunto(s)
Ataxia Telangiectasia , Oryzias , Animales , Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Oryzias/genética , Oryzias/metabolismo , Mutación de Línea Germinal , Proteínas Supresoras de Tumor/genética , Daño del ADN , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Mutación , Carcinogénesis , Transformación Celular Neoplásica , Fenotipo
2.
Glycoconj J ; 40(3): 315-322, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36933118

RESUMEN

It has been clarified that pathogens bind to glycosphingolipid (GSL) receptors in mammals, but there have been very few reports on pathogen-binding GSLs in fish. Vibrios are facultative anaerobic bacteria ubiquitous in marine and brackish environments. They are members of the normal intestinal microflora of healthy fish, but some species can cause a disease called vibriosis in fish and shellfish when the hosts are physiologically or immunologically weakened. The adherence of vibrios to host intestinal tracts is a significant event not only for survival and growth but also in terms of pathogenicity. We show in this mini-review that sialic acid-containing GSLs (gangliosides), GM4 and GM3, are receptors to which vibrios adhere to epithelial cells in the intestinal tract of fish. We also describe the enzymes responsible for synthesizing these Vibrio-binding gangliosides in fish.


Asunto(s)
Gangliósidos , Vibrio , Animales , Gangliósidos/metabolismo , Glicoesfingolípidos/metabolismo , Intestinos , Peces/metabolismo , Vibrio/metabolismo , Mamíferos/metabolismo
4.
Environ Pollut ; 268(Pt B): 115957, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33158613

RESUMEN

Many studies using experimental and wild animals have reported negative effects of microplastic beads (MPs) ingestion. However, data regarding the lowest observed adverse effect levels (LOAELs) of MPs remain limited. Our aim was to evaluate the adverse effect levels of polyethylene MPs (10-63 µm diameter) with respect to growth, reproduction, and the eyes and kidneys of medaka (Oryzias latipes) under breeding conditions to contribute to future research involving LOAEL determinations. Fish were exposed to 0.009 mg-MPs (approximately 1000 particles)/L to 0.32 mg-MPs (approximately 40,000 particles)/L for 12 weeks. The eyes and kidneys were evaluated by histopathologic analysis. Although histologic analyses indicated an absence of MPs in the tissues, the eyes and kidneys as well as reproduction were adversely affected by increasing MP concentrations. The number of spawned eggs decreased, and changes were noted in the eyes of fish exposed to ≥0.032 mg-MPs/L under breeding conditions. The eyes exhibited thinning of the optic nerve fiber layer and dilatation of retinal capillaries compared with medaka not treated with MPs. Changes in the kidneys were observed in fish exposed to ≥0.065 mg-MPs/L. The mesangial matrix in the glomerulus of the kidneys was expanded compared with non-treated medaka, suggesting a deterioration in renal function. Analyses of an oxidative stress marker in the tissues indicated that lesion progression was associated with increased oxidative stress. Furthermore, a comparison of adverse effect levels suggested that MPs were more toxic to the eyes and reproduction than the kidneys or growth. Our data should prove useful for determining the LOAELs of polyethylene beads on vertebrates and enhance understanding of the mechanism underlying the biological toxicity of polyethylene MPs.


Asunto(s)
Oryzias , Contaminantes Químicos del Agua , Animales , Riñón , Microesferas , Plásticos , Polietileno/toxicidad , Reproducción
5.
J Biochem ; 168(3): 213-222, 2020 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-32251518

RESUMEN

Transglutaminases are an enzyme family that catalyses protein cross-linking essential for several biological functions. In the previous studies, we characterized the orthologues of the mammalian transglutaminase family in medaka (Oryzias latipes), an established fish model. Among the human isozymes, tissue-type transglutaminase (TG2) has multiple functions that are involved in several biological phenomena. In this study, we established medaka mutants deficient for the orthologue of human TG2 using the CRISPR/Cas9 and transcription activator-like effector nucleases systems. Although apparent morphological changes in the phenotype were not observed, movement retardation was found in the mutant fish when evaluated by a tank-diving test. Furthermore, comparative immunohistochemistry analysis using in this fish model revealed that orthologue of human TG2 was expressed at the periventricular layer of the optic tectum. Our findings provide novel insight for the relationship between tissue-type transglutaminase and the nervous system and the associated behaviour.


Asunto(s)
Proteínas de Peces/genética , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Movimiento , Oryzias/genética , Oryzias/metabolismo , Transglutaminasas/genética , Transglutaminasas/metabolismo , Animales , Animales Modificados Genéticamente , Conducta Animal , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Edición Génica , Humanos , Fenotipo , Proteína Glutamina Gamma Glutamiltransferasa 2
6.
J Toxicol Pathol ; 32(4): 297-303, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31719758

RESUMEN

The aim of this study was to elucidate the renal lesions of leptin receptor-deficient medaka showing hyperglycemia and hypoinsulinemia and to evaluate the usefulness of the medaka as a model of diabetic nephropathy. Leptin receptor-deficient medaka at 20 and 30 weeks of age showed hyperglycemia and hypoinsulinemia; they also showed a higher level of plasma creatinine than the control medaka. Histopathologically, dilation of glomerular capillary lumina and of afferent/efferent arterioles was observed in leptin receptor-deficient medaka at 20 weeks of age, and then glomerular enlargement with cell proliferation and matrix expansion, formation of fibrin cap-like lesions, glomerular atrophy with Bowman's capsule dilation, and renal tubule dilation were observed at 30 weeks of age. These histopathological characteristics of leptin receptor-deficient medaka were similar to the characteristics of kidney lesions of human and rodent models of type II diabetes mellitus, making leptin receptor-deficient medaka a useful model of diabetic nephropathy.

7.
Environ Pollut ; 254(Pt B): 113094, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31479815

RESUMEN

Research using various species of wild and cultured fish has identified negative effects of short-term exposure to microbeads. Although wild animals might be contaminated with microbeads and/or other pharmaceuticals, data regarding the long-term effects remain limited. To clearly elucidate the effects of microbeads, studies of long-term exposure using animal models are necessary. Our aim was to elucidate the effects of microbeads alone on the growth and fecundity of medaka following long-term exposure (12 weeks). In experiment 1, fish groups (except controls) were temporarily exposed to polyethylene microbeads (10-63 µm diameter) a low dose of 0.065 microbeads-mg/L and high dose of 0.65 microbeads-mg/L. In experiment 2, see-through medaka and fluorescent polyethylene microbeads (10-45 µm diameter) were used to estimate the retention time of ingested microbeads in the digestive tract, which was 4-9 days. The low dose of microbeads did not affect growth but did decrease the number of eggs and the hatching rate. The high dose decreased growth, the number of eggs, and hatching rate. Growth differences were recognized for the first time at 7 weeks, and differences in the number of eggs at 12 weeks. Thus, long-term tests using medaka indicated that microbeads per se exhibit growth inhibition and reproductive toxicity. These effects could be associated with nutritional factors resulting from the long retention time of microbeads in the digestive tract. We also determined the dose that affects only fecundity. This suggests that normal growth of medaka in the wild does not mean the environment is free from microbead contamination. We are thus attempting to identify new biological indexes for monitoring the status of microbead contamination using our system.


Asunto(s)
Microesferas , Oryzias/fisiología , Polietileno/toxicidad , Contaminantes Químicos del Agua/toxicidad , Animales , Femenino , Intestinos/patología , Masculino , Oryzias/crecimiento & desarrollo , Reproducción/efectos de los fármacos , Pruebas de Toxicidad
8.
Artículo en Inglés | MEDLINE | ID: mdl-29567411

RESUMEN

Lamin is an intermediate protein underlying the nuclear envelope and it plays a key role in maintaining the integrity of the nucleus. A defect in the processing of its precursor by a metalloprotease, ZMPSTE24, results in the accumulation of farnesylated prelamin in the nucleus and causes various diseases, including Hutchinson-Gilford progeria syndrome (HGPS). However, the role of lamin processing is unclear in fish species. Here, we generated zmpste24-deficient medaka and evaluated their phenotype. Unlike humans and mice, homozygous mutants did not show growth defects or lifespan shortening, despite lamin precursor accumulation. Gonadosomatic indices, blood glucose levels, and regenerative capacity of fins were similar in 1-year-old mutants and their wild-type (WT) siblings. Histological examination showed that the muscles, subcutaneous fat tissues, and gonads were normal in the mutants at the age of 1 year. However, the mutants showed hypersensitivity to X-ray irradiation, although p53target genes, p21 and mdm2, were induced 6 h after irradiation. Immunostaining of primary cultured cells from caudal fins and visualization of nuclei using H2B-GFP fusion proteins revealed an abnormal nuclear shape in the mutants both in vitro and in vivo. The telomere lengths were significantly shorter in the mutants compared to WT. Taken together, these results suggest that zmpste24-deficient medaka phenocopied HGPS only partially and that abnormal nuclear morphology and lifespan shortening are two independent events in vertebrates.


Asunto(s)
Núcleo Celular/patología , Modelos Animales de Enfermedad , Proteínas de Peces/deficiencia , Proteínas de la Membrana/deficiencia , Metaloendopeptidasas/deficiencia , Oryzias/genética , Progeria/patología , Aletas de Animales/enzimología , Aletas de Animales/patología , Aletas de Animales/efectos de la radiación , Animales , Animales Modificados Genéticamente , Núcleo Celular/enzimología , Núcleo Celular/efectos de la radiación , Forma del Núcleo Celular/efectos de la radiación , Células Cultivadas , Codón sin Sentido , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Técnicas de Inactivación de Genes , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Oryzias/metabolismo , Progeria/enzimología , Progeria/genética , Tolerancia a Radiación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Supervivencia , Acortamiento del Telómero/efectos de la radiación
9.
J Toxicol Pathol ; 31(1): 65-72, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-29479143

RESUMEN

Ocular lesions in leptin receptor-deficient medaka were examined histopathologically at 10, 28, and 37 weeks post hatching. Leptin receptor-deficient medaka at 28 and 37 weeks old showed hyperglycemia and hypoinsulinemia. Histopathologically, vacuolation, swelling, fragmentation, and liquefaction of the lens fibers and dilatation of the retinal central veins, retinal capillaries, iridal veins and capillaries, and choroidal veins were observed in leptin receptor-deficient medaka at 28 and 37 weeks old. Thinning of the total retina, pigment epithelial layer, layer of rods and cones, outer granular layer, outer plexiform layer, inner granular layer, and inner plexiform layer was observed in leptin receptor-deficient medaka at 28 and 37 weeks compared with in control medaka. These histopathological characteristics in leptin receptor-deficient medaka are similar to characteristics in ocular lesions of rodent models for type II diabetes mellitus, making leptin receptor-deficient medaka a useful model of diabetic cataract and retinopathy.

10.
Gen Comp Endocrinol ; 195: 9-20, 2014 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-24505600

RESUMEN

The first studies that identified leptin and its receptor (LepR) in mammals were based on mutant animals that displayed dramatic changes in body-weight and regulation of energy homeostasis. Subsequent studies have shown that a deficiency of leptin or LepR in homoeothermic mammals results in hyperphagia, obesity, infertility and a number of other abnormalities. The physiological roles of leptin-mediated signaling in ectothermic teleosts are still being explored. Here, we produced medaka with homozygous LepR gene mutation using the targeting induced local lesions in a genome method. This knockout mutant had a point mutation of cysteine for stop codon at the 357th amino acid just before the leptin-binding domain. The evidence for loss of function of leptin-mediated signaling in the mutant is based on a lack of response to feeding in the expression of key appetite-related neuropeptides in the diencephalon. The mutant lepr−/− medaka expressed constant up-regulated levels of mRNA for the orexigenic neuropeptide Ya and agouti-related protein and a suppressed level of anorexigenic proopiomelanocortin 1 in the diencephalon independent of feeding, which suggests that the mutant did not possess functional LepR. Phenotypes of the LepR-mutant medaka were analyzed in order to understand the effects on food intake, growth, and fat accumulation in the tissues. The food intake of the mutant medaka was higher in post-juveniles and adult stages than that of wild-type (WT) fish. The hyperphagia led to a high growth rate at the post-juvenile stage, but did not to significant alterations in final adult body size. There was no additional deposition of fat in the liver and muscle in the post-juvenile and adult mutants, or in the blood plasma in the adult mutant. However, adult LepR mutants possessed large deposits of visceral fat, unlike in the WT fish, in which there were none. Our analysis confirms that LepR in medaka exert a powerful influence on the control on food intake. Further analyses using the mutant will contribute to a better understanding of the role of leptin in fish. This is the first study to produce fish with leptin receptor deficiency.


Asunto(s)
Animales Modificados Genéticamente/crecimiento & desarrollo , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/fisiología , Técnicas de Inactivación de Genes , Grasa Intraabdominal/efectos de los fármacos , Neuropéptidos/farmacología , Receptores de Leptina/fisiología , Proteína Relacionada con Agouti/metabolismo , Animales , Animales Modificados Genéticamente/metabolismo , Apetito/efectos de los fármacos , Apetito/fisiología , Diencéfalo/efectos de los fármacos , Diencéfalo/metabolismo , Ingestión de Alimentos/efectos de los fármacos , Hiperfagia/genética , Hiperfagia/patología , Leptina/metabolismo , Mutación/genética , Obesidad/metabolismo , Oryzias/genética , Oryzias/crecimiento & desarrollo , Oryzias/metabolismo , Regulación hacia Arriba
11.
Fish Physiol Biochem ; 40(2): 511-25, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24026769

RESUMEN

Bile acid transporters belonging to the SLC10A protein family, Na+ taurocholate cotransporting polypeptide (NTCP or SLC10A1), apical sodium-dependent bile salt transporter (ASBT or SLC10A2), and organic solute transporter alpha (Ost-alpha) have been known to play critical roles in the enterohepatic circulation of bile acids in mammals. In this study, ntcp, asbt, and ost-alpha-1/-2 cDNA were cloned, their tissue distributions were characterized, and the effects of fasting and bile acid administration on their expression were examined in rainbow trout Oncorhynchus mykiss. The structural characteristics of Ntcp, Asbt, and Ost-alpha were well conserved in trout, and three-dimensional structure analysis showed that Ntcp and Asbt were similar to each other. Tissue distribution analysis revealed that trout asbt was primarily expressed in the hindgut, while ntcp expression occurred in the brain, and ost-alpha-1/-2 was mainly expressed in the liver or ovary. Although asbt and ost-alpha-1 mRNA levels in the gut increased in response to fasting for 4 days, ost-alpha-1 expression in the liver decreased. Similarly, bile acid administration increased asbt and ost-alpha-1 expression levels in the gut, while those of ntcp and ost-alpha-2 in the liver decreased. These results suggested that the genes asbt, ntcp, and ost-alpha are involved in bile acid transport in rainbow trout.


Asunto(s)
Ácidos y Sales Biliares/administración & dosificación , Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/genética , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/química , Clonación Molecular , ADN Complementario/genética , Ayuno/metabolismo , Femenino , Proteínas de Peces/química , Expresión Génica , Masculino , Glicoproteínas de Membrana/química , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Distribución Tisular
12.
BMC Genomics ; 14: 786, 2013 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-24225309

RESUMEN

BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.


Asunto(s)
Cruzamiento , Mutagénesis , Genética Inversa , Takifugu/genética , Alelos , Animales , Codón sin Sentido/efectos de los fármacos , Codón sin Sentido/genética , Etilnitrosourea/administración & dosificación , Femenino , Genoma/efectos de los fármacos , Masculino
13.
Artículo en Inglés | MEDLINE | ID: mdl-23872320

RESUMEN

In mammals, cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) are rate-limiting enzymes in bile acid synthesis. In addition, a small heterodimer partner (SHP) is also known to inhibit bile acid synthesis via the suppression of CYP7A1 and CYP8B1 expression. However, little information is currently available regarding primary structure of the genes involved in bile acid synthesis in fish. We therefore cloned cyp7a1, cyp8b1 and shp genes from rainbow trout and obtained cDNAs encoding two isoforms each of Cyp7a1 (-1 and -2), Cyp8b1 (-1 and -2) and Shp (-1 and -2). Both cyp7a1-1 and -2 encoded proteins of 512 amino acids. Trout cyp7a1-1 was expressed not only primarily in the kidney, pyloric caecum and mid-gut, but also weakly in the liver, eye, gill and ovary. cyp7a1-2 was highly expressed in the liver, pyloric caecum and mid-gut. cyp8b1-1 and -2, which encoded proteins of 512 and 509 amino acids, respectively, were principally expressed in the liver. Both shp-1 and -2, which encoded proteins of 288 and 290 amino acids, respectively, were strongly expressed in the liver, but shp-2 was also highly expressed in the gallbladder and digestive tract. The temporal changes in the expression of cyp7a1-1/-2, cyp8b1-1/-2 and shp-1/-2 in the liver were assessed after consumption of a single meal. Expression of cyp7a1-1/-2 and cyp8b1-1/-2 increased within 3h post feeding (hpf) when the stomach was still approximately 84% full and the gallbladder was almost completely empty. Although the expression of shp-1 did not change after feeding, the expression pattern of shp-2 was inversely related to the expression patterns of cyp7a1-1/-2 and cyp8b1-1/-2. Specifically, shp-2 expression decreased until 3 hpf before returning to initial levels at 24 hpf. These findings suggest that Cyp7a1s/8b1s and Shp-2 function antagonistically in bile acid synthesis in rainbow trout.


Asunto(s)
Colesterol 7-alfa-Hidroxilasa/metabolismo , Proteínas de Peces/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Esteroide 12-alfa-Hidroxilasa/metabolismo , Secuencia de Aminoácidos , Animales , Ácidos y Sales Biliares/biosíntesis , Colesterol 7-alfa-Hidroxilasa/química , Colesterol 7-alfa-Hidroxilasa/genética , Proteínas de Peces/química , Proteínas de Peces/genética , Vesícula Biliar/enzimología , Expresión Génica , Intestinos/enzimología , Hígado/enzimología , Datos de Secuencia Molecular , Oncorhynchus mykiss , Especificidad de Órganos , Filogenia , Periodo Posprandial , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Esteroide 12-alfa-Hidroxilasa/química , Estómago/enzimología
14.
FEMS Microbiol Lett ; 341(1): 18-26, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23320941

RESUMEN

Vibrios, distributed in marine and brackish environments, can cause vibriosis in fish and shellfish under appropriate conditions. Previously, we clarified by thin-layer chromatography (TLC) overlay assay that (35)S-labeled Vibrio trachuri adhered to GM4 isolated from red sea bream intestine. However, whether GM4 actually functions on epithelial cells as an attachment site for vibrios still remains to be uncovered. We found that six isolates, classified as V. harveyi, V. campbellii, and V. splendidus, from intestinal microflora of red sea bream adhered to GM4 but not galactosylceramide (GalCer) by TLC-overlay assay. Tissue-overlay assays revealed that V. harveyi labeled with green fluorescent protein (GFP) adhered to epithelial cells of red sea bream intestine where GM4 and GalCer were found to be distributed on the top layer of actin filaments by immunohistochemical analysis using corresponding antibodies. The number of adhering vibrios was diminished by pretreatment with anti-GM4 antibody, but not anti-GalCer antibody. These results clearly indicate that vibrios adhere to epithelial cells of red sea bream intestine utilizing GM4 as an attachment site.


Asunto(s)
Adhesión Bacteriana , Células Epiteliales/microbiología , Gangliósidos/metabolismo , Dorada/microbiología , Vibrio/patogenicidad , Citoesqueleto de Actina/metabolismo , Animales , Anticuerpos/metabolismo , Carga Bacteriana , Sitios de Unión , Cromatografía en Capa Delgada , Galactosilceramidas/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Intestinos/citología , Intestinos/microbiología , Vibrio/clasificación , Vibrio/aislamiento & purificación , Vibrio/metabolismo
15.
Dev Biol ; 359(1): 82-94, 2011 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-21925159

RESUMEN

Myostatin (MSTN) functions as a negative regulator of skeletal muscle mass. In mammals, MSTN-deficient animals result in an increase of skeletal muscle mass with both hyperplasia and hypertrophy. A MSTN gene is highly conserved within the fish species, allowing speculation that MSTN-deficient fish could exhibit a double-muscled phenotype. Some strategies for blocking or knocking down MSTN in adult fish have been already performed; however, these fish show either only hyperplastic or hypertrophic growth in muscle fiber. Therefore, the role of MSTN in fish myogenesis during post-hatch growth remains unclear. To address this question, we have made MSTN-deficient medaka (mstnC315Y) by using the targeting induced local lesions in a genome method. mstnC315Y can reproduce and have the same survival period as WT medaka. Growth rates of WT and mstnC315Y were measured at juvenile (1-2wk post-hatching), post-juvenile (3-7wk post-hatching) and adult (8-16wk post-hatching) stages. In addition, effects of MSTN on skeletal muscle differentiation were investigated at histological and molecular levels at each developmental stage. As a result, mstnC315Y show a significant increase in body weight from the post-juvenile to adult stage. Hyper-morphogenesis of skeletal muscle in mstnC315Y was accomplished due to hyperplastic growth from post-juvenile to early adult stage, followed by hypertrophic growth in the adult stage. Myf-5 and MyoD were up-regulated in mstnC315Y at the hyperplastic growth phase, while myogenin was highly expressed in mstnC315Y at the hypertrophic growth phase. These indicated that MSTN in medaka plays a dual role for muscle fiber development. In conclusion, MSTN in medaka regulates the number and size of muscle fiber in a temporally-controlled manner during posthatch growth.


Asunto(s)
Músculo Esquelético/crecimiento & desarrollo , Miostatina/genética , Oryzias/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Clonación Molecular , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Hiperplasia , Hipertrofia , Inmunohistoquímica , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Oryzias/genética , Fenotipo , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido
16.
J Biol Chem ; 284(44): 30534-46, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19542236

RESUMEN

We have previously reported that fish pathogens causing vibriosis specifically adhere to GM4 on the epithelial cells of fish intestinal tracts (Chisada, S., Horibata, Y., Hama, Y., Inagaki, M., Furuya, N., Okino, N., and Ito, M. (2005) Biochem. Biophys. Res. Commun. 333, 367-373). To identify the gene encoding the enzyme for GM4 synthesis in the fish intestinal tract, a phylogenetic tree of vertebrate ST3GalVs, including Danio rerio and Oryzias latipes, was generated in which two putative subfamilies of fish ST3GalVs were found. Two putative ST3GalVs of zebrafish (zST3GalV-1 and -2), each belonging to different subfamilies, were cloned from the zebrafish cDNA library. Interestingly, zST3GalV-1 synthesized GM3 (NeuAcalpha2-3Galbeta1-4Glcbeta1-1'Cer) but not GM4, whereas zSTGalV-2 synthesized both gangliosides in vitro when expressed in CHO-K1 and RPMI1846 cells. Flow cytometric analysis using anti-GM4 antibody revealed that the transformation of RPMI1846 cells with zST3GalV-2 but not zST3GalV-1 cDNA increased the cell-surface expression of GM4. Whole mount in situ hybridization showed that the zST3GalV-2 transcript was strongly expressed in the gastrointestinal tract, whereas zST3GalV-1 was expressed in the brain and esophagus but not gastrointestinal tract in 3-day post-fertilization embryos. It has long been a matter of controversy which enzyme is responsible for the synthesis of GM4 in mammals. We found that three isoforms of mouse ST3GalV (mST3GalV) having different N-terminal sequences can synthesize GM4 as well as GM3 when expressed in RPMI1846 and CHO-K1 cells. Furthermore, mST3GalV knock-out mice were found to lack GM4 synthase activity and GM4 in contrast to wild-type mice. These results clearly indicate that zST3GalV-2 and mST3GalV are the enzymes responsible for the synthesis of GM4 in zebrafish and mice, respectively.


Asunto(s)
Gangliósidos/biosíntesis , Filogenia , Sialiltransferasas/metabolismo , Animales , Clonación Molecular , ADN Complementario , Embrión de Mamíferos , Ratones , Ratones Noqueados , ARN Mensajero/análisis , Sialiltransferasas/genética , Distribución Tisular , Pez Cebra , beta-Galactosida alfa-2,3-Sialiltransferasa
17.
Comp Biochem Physiol B Biochem Mol Biol ; 147(4): 635-44, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17499534

RESUMEN

First, we attempted to isolate glycosphingolipids from eel serum HDL. A single ganglioside containing N-acetylneuraminic acid (NeuAc), which is positive with resorcinol and orcinol reactions, was purified. The mobilities of the purified ganglioside and its lyso-form on high performance TLC were similar as those of authentic GM4 and its lyso-form, respectively. The mass of the purified ganglioside was determined by TOF mass spectrometer, and the mass of its oligosaccharide was the same as that of authentic GM4 from human brain consisting of disaccharide of NeuAc and galactose. The ganglioside from eel HDL was not hydrolyzed by recombinant endoglycoceramidase II, which cannot hydrolyze between galactose and ceramide of gangliosides, but hydrolyzes between glucose and ceramide. We concluded from these results that the ganglioside purified from eel serum HDL is GM4. Second, we investigated the effects of the ganglioside on binding of HDL labeled with fluorescein isothiocyanate (FITC-HDL) to cultured eel hepatocytes and on FITC-HDL ligand blotting by using plasma membrane proteins of the hepatocytes. Stimulatory effect of GM4 on FITC-HDL binding to the hepatocytes and FITC-HDL ligand blotting suggests strongly that GM4 is a ligand for HDL binding protein of eel hepatocytes.


Asunto(s)
Proteínas Portadoras/metabolismo , Anguilas/sangre , Gangliósidos/sangre , Gangliósidos/fisiología , Lipoproteínas HDL/química , Proteínas de Unión al ARN/metabolismo , Animales , Anticuerpos/farmacología , Apolipoproteína A-I/inmunología , Apolipoproteína A-II/inmunología , Bovinos , Células Cultivadas , Anguilas/metabolismo , Femenino , Gangliósido G(M1)/farmacología , Gangliósidos/metabolismo , Gangliósidos/farmacología , Hepatocitos/metabolismo , Humanos , Ligandos , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Modelos Biológicos , Unión Proteica , Conejos
18.
Biochem Biophys Res Commun ; 333(2): 367-73, 2005 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-15979459

RESUMEN

Three major glycosphingolipids (tentatively designated IGL-1, 2, and 3) were isolated from the intestine of red sea bream (Pagrus major) and were subjected to a TLC-overlay assay with (35)S-labeled Vibrio trachuri which causes vibriosis of fish. The bacteria adhered to IGL-2, which was determined to be a GM4 ganglioside (NeuAcalpha2-3Galbeta1-ceramide). The fatty acid portion of IGL-2 was composed of 2-hydroxy C22:0, C24:0, and C24:1, in addition to the non-hydroxy C16:0 and C18:0, while the sphingoid base was composed exclusively of sphingenine (d18:1). Among glycosphingolipids tested, V. trachuri adhered to GM4 the most strongly followed by adherence to GM3 and GalCer, but the bacteria did not adhere to GM1a, GM2, LacCer, or GlcCer. V. trachuri was found to aggregate with the erythrocytes coated with GM4, but not with those coated with GM1a or GM2, thus indicating that specific adhesion occurs on intact cells. Interestingly, the dynamics for adhesion of V. trachuri to glycosphingolipids was defined by the structure of not only the sugar moiety but also the ceramide moiety, since the bacteria adhered to GM4 which contained 2-hydroxy fatty acids much more strongly than to that which contained non-hydroxy fatty acids.


Asunto(s)
Adhesión Bacteriana/fisiología , Peces/metabolismo , Gangliósidos/química , Gangliósidos/metabolismo , Glicoesfingolípidos/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/microbiología , Vibrio/fisiología , Animales , Adhesión Celular/fisiología , Ácidos Grasos/química , Peces/microbiología , Hidroxiácidos/química , Proteínas de la Membrana/metabolismo
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