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1.
Poult Sci ; 101(5): 101804, 2022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35325830

RESUMEN

Dietary supplementation of green tea powder (GTP) changes egg quality of hens, however, whether these changes affect incubation is still unknown. This study was to compare the proteomic difference of incubated eggs from hens with GTP supplemented or not. Huainan partridge chickens (1,080) at 35 wk of age were allocated into 2 groups, one group fed basal diet (CG) and one group fed basal diet plus 1% GTP (EG). After 4 wk feeding, artificially fertilized eggs were collected for yolk cholesterol determination and incubation. During incubation, 6 embryos from each group were randomly selected in each day for yolk protein extraction and quantification. Yolk cholesterol content was significantly lower, while the hatchability was significantly higher in EG than that of the CG group (P < 0.05). Yolk protein concentration at embryonic days (ED) of 0, 2, 6, and 13 showed significant changes and were selected for proteomic analysis by 2-dimensional gel electrophoresis combined with liquid chromatography-tandem mass spectrometry. Fifty-one differentially expressed (DE) protein spots were identified among different incubation stages between CG and EG group which were mainly classified into vitellogenin, immunoglobulin, and ovoinhibitor, and occupied 45.1, 23.5, and 15.7%, respectively, to the total DE proteins. Ovotransferrin, participated in extracellular sequestering of iron ion process, was significantly lower in EG group than that of the CG group (P < 0.05). Ig light chain precursor (Immunoglobulin) exhibited higher expression at ED6 in EG group as compared with that of the CG group, and was participated in immune response related processes. Ovoinhibitor, mainly involved in protease binding activity, showed lower abundance at ED13 in EG group as compared with that of the CG group. Vitellogenin-3, showed lower expression in EG group as compared with that of the CG group, was mainly participated in lipid transportation and localization according to GO enrichment. Chickens fed diet with GTP provided eggs more antioxidant ability that increased hatchability, indicated that GTP could be considered as additive in breeding layer.


Asunto(s)
Antioxidantes , Pollos , Alimentación Animal/análisis , Animales , Antioxidantes/análisis , Colesterol/análisis , Dieta/veterinaria , Proteínas del Huevo/análisis , Yema de Huevo/química , Femenino , Guanosina Trifosfato/análisis , Polvos/análisis , Proteómica , , Vitelogeninas/análisis
2.
Anal Biochem ; 424(1): 8-11, 2012 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-22369893

RESUMEN

An aptamer-molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500 nM with a detection limit of 0.1 nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.


Asunto(s)
Adenosina Trifosfato/análisis , Aptámeros de Nucleótidos/metabolismo , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Animales , Bovinos , Citidina Trifosfato/análisis , Guanosina Trifosfato/análisis , Albúmina Sérica Bovina/análisis , Espectrometría de Fluorescencia , Uridina Trifosfato/análisis
3.
Anal Chem ; 79(3): 1158-63, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17263349

RESUMEN

We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.


Asunto(s)
Electroforesis Capilar/métodos , GTP Fosfohidrolasas/análisis , Receptores Acoplados a Proteínas G , Línea Celular , Membrana Celular/enzimología , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/análisis , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/análisis , Guanosina Difosfato/biosíntesis , Guanosina Trifosfato/análisis , Guanosina Trifosfato/metabolismo , Humanos , Ligandos , Receptores Adrenérgicos alfa 2 , Transfección
4.
Exp Cell Res ; 253(2): 474-82, 1999 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-10585270

RESUMEN

Human 293 cells were stably transfected with a plasmid introducing a receptor for the ecdysone analog muristerone. The cells were further stably transfected with muristerone-inducible expression vectors carrying either the cDNA for the human high K(M) 5'-nucleotidase or the coding sequence of the nucleotidase linked to the 5'-end of the sequence for the green fluorescent protein. Upon induction, both types of transfectants overproduced nucleotidase activity in a time- and dose-dependent manner. Western blots gave values close to the expected subunit molecular masses of 65 and 92 kDa, respectively, excluding processing of the induced proteins. Cells induced to overexpress the nucleotidase showed a decreased growth rate and contained smaller pools of each of the four common ribonucleoside triphosphates. They showed no increased resistance to the toxicity of 2-chlorodeoxyadenosine.


Asunto(s)
5'-Nucleotidasa/genética , Regulación Enzimológica de la Expresión Génica/fisiología , 5'-Nucleotidasa/análisis , 5'-Nucleotidasa/metabolismo , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Antineoplásicos/farmacología , Western Blotting , Células Cultivadas , Cladribina/farmacología , Clonación Molecular , Citidina Trifosfato/análisis , Citidina Trifosfato/metabolismo , ADN Complementario , Ecdisterona/análogos & derivados , Ecdisterona/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Proteínas Fluorescentes Verdes , Guanosina Trifosfato/análisis , Guanosina Trifosfato/metabolismo , Humanos , Indicadores y Reactivos/metabolismo , Riñón/citología , Proteínas Luminiscentes/genética , Plásmidos , Receptores de Esteroides/genética , Transfección , Uridina Trifosfato/análisis , Uridina Trifosfato/metabolismo
5.
Neurobiol Aging ; 19(5): 427-35, 1998.
Artículo en Inglés | MEDLINE | ID: mdl-9880045

RESUMEN

Galanin (GAL) has been proposed to be an inhibitory modulator of cholinergic memory pathways because it acts within the hippocampus to inhibit the release and antagonize the postsynaptic actions of acetylcholine. Here we have used: 1) slice binding and quantitative autoradiography to assess the density and occupancy of GAL receptors; and 2) in situ hybridization histochemistry to assess expression of the GALR1 receptor subtype in the ventral hippocampus of 3-month-old and 21-month-old Fischer 344 male rats. We detected a small but significant (p < or = 0.0003) age-related reduction in 125I-GAL binding-site density in the ventral hippocampus and entorhinal cortex under standard binding conditions. Post-hoc analysis indicated that this reduction with age persisted in the CA1 radiatum and entorhinal cortex following GTP-induced desaturation to unmask pre-existent GAL receptors occupied by endogenous ligand. It was not associated with a significant change in peak GALR1 gene expression in the hippocampus. Because a portion of GAL receptors in this region have been postulated to function as presynaptic auto-receptors on cholinergic fiber terminals, the reduction in GAL binding sites with age may be a consequence of age-related alterations in GAL receptor expression by basal forebrain cholinergic neurons which project to the ventral hippocampus.


Asunto(s)
Envejecimiento/fisiología , Corteza Entorrinal/química , Hipocampo/química , Receptores de Neuropéptido/análisis , Animales , Ventrículos Cerebrales/química , ADN Complementario , Proteínas de Unión al GTP/análisis , Expresión Génica/fisiología , Guanosina Trifosfato/análisis , Hibridación in Situ , Radioisótopos de Yodo , Masculino , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Receptores de Galanina , Receptores de Neuropéptido/genética
6.
Ann Emerg Med ; 22(9): 1385-91, 1993 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-8363112

RESUMEN

STUDY OBJECTIVE: To evaluate the effects of standard-dose versus high-dose epinephrine on myocardial high-energy phosphate metabolism during resuscitation from cardiac arrest. DESIGN: Prospective, nonrandomized, controlled study using a swine model of cardiac arrest and resuscitation. INTERVENTIONS: After anesthesia, intravascular pressure instrumentation, and ten minutes of ventricular fibrillation arrest, closed-chest CPR was begun. After three minutes of CPR, animals were allocated to receive either 0.02 mg/kg i.v. standard-dose epinephrine (eight) or 0.2 mg/kg i.v. high-dose epinephrine (nine). The animals underwent thoracotomy and rapid-freezing transmural myocardial core biopsy for high-energy phosphate analysis 3.5 minutes after epinephrine administration. High-energy phosphate values were blindly determined using high-pressure liquid chromatography. RESULTS: Intravascular pressure (mm Hg) and high-energy phosphate (nmol/mg protein) results for standard-dose epinephrine versus high-dose epinephrine are, respectively, coronary perfusion pressure, 15.3 +/- 7.8 versus 23.7 +/- 5.5 (P = .0009); phosphocreatine, 0.4 +/- 0.8 versus 6.2 +/- 4.4 (P = .0003); adenosine triphosphate, 9.8 +/- 4.8 versus 12.7 +/- 5.7 (P = .30); adenosine diphosphate, 5.4 +/- 2.1 versus 6.1 +/- 1.3 (P = .41); and adenylate charge, 0.68 +/- 0.12 versus 0.72 +/- 0.12 (P = .87). CONCLUSION: High-dose epinephrine does not deplete myocardial high-energy phosphate when given in this model of prolonged ventricular fibrillation. High-dose epinephrine increases coronary perfusion pressure compared with standard-dose epinephrine. High-dose epinephrine administration repletes phosphocreatine during closed-chest CPR, thereby increasing myocardial energy stores.


Asunto(s)
Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Reanimación Cardiopulmonar/métodos , Epinefrina/farmacología , Paro Cardíaco/tratamiento farmacológico , Hemodinámica/efectos de los fármacos , Miocardio/química , Miocardio/metabolismo , Fosfocreatina/análisis , Porcinos , Fibrilación Ventricular/tratamiento farmacológico , Adenosina/análisis , Adenosina Monofosfato/análisis , Animales , Biopsia , Análisis de los Gases de la Sangre , Cromatografía Líquida de Alta Presión , Protocolos Clínicos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Epinefrina/administración & dosificación , Guanosina Trifosfato/análisis , Paro Cardíaco/metabolismo , Paro Cardíaco/patología , Paro Cardíaco/fisiopatología , Inyecciones Intravenosas , Inosina/análisis , Inosina Monofosfato/análisis , Miocardio/patología , Fibrilación Ventricular/metabolismo , Fibrilación Ventricular/patología , Fibrilación Ventricular/fisiopatología
7.
J Biol Chem ; 265(31): 19137-42, 1990 Nov 05.
Artículo en Inglés | MEDLINE | ID: mdl-2229067

RESUMEN

The cap structure in human U6 small nuclear (sn)RNA, gamma-monomethylguanosine triphosphate (meGTP), was conjugated to human serum albumin and used as antigen to raise polyclonal antibodies in rabbits. The resulting antibodies reacted specifically with meGTP but not with GTP, GDP, GMP, meGMP, meATP, meCTP, meUTP, or with methyl phosphate in enzyme-linked immunosorbent assay and/or in radioimmunoassays. Although less efficiently, meGDP was also recognized by these antibodies. Indirect immunofluorescence studies with anti-meGTP antibodies showed predominantly nuclear immunofluorescence. Anti-meGTP antibodies immunoprecipitated intact U6 snRNA from a mixture of HeLa cell RNAs. In addition to the U6 snRNA, anti-meGTP antibodies immunoprecipitated several additional small RNAs that varied in length from approximately 50 to 330 nucleotides. These RNAs contained the meGTP cap structure and are structurally distinct from U6 snRNA. One of these meGTP-containing RNAs was found to be previously characterized 7SK RNA; human 7SK RNA synthesized in vitro also contained the same cap structure. Results obtained in this study provide evidence for the presence of gamma-monomethyl-GTP cap structure in a wide spectrum of human cellular RNAs. These antibodies will be useful in studying the structure and function of this new family of small RNAs.


Asunto(s)
Anticuerpos , Guanosina Trifosfato/análogos & derivados , Caperuzas de ARN/análisis , ARN Nuclear Pequeño/análisis , Fabaceae/análisis , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/análisis , Guanosina Trifosfato/metabolismo , Células HeLa/química , Humanos , Peso Molecular , Plantas Medicinales , Caperuzas de ARN/inmunología , ARN Nuclear Pequeño/inmunología , Uridina Trifosfato/metabolismo
8.
Biochem Biophys Res Commun ; 135(2): 549-55, 1986 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-3485974

RESUMEN

Phosphorus nuclear magnetic resonance spectra of the Ha-ras oncogene product p21 and its nucleotide complexes have been obtained. It is shown that the 31P nuclear magnetic resonance spectra of a number of nucleotide-enzyme complexes show some common features. In particular, the chemical shift values of the beta-phosphorus resonance of enzyme-bound NTP and NDP (N = A, G) of hydrolases exhibit a downfield shift virtually identical for myosin, elongation factor Tu, and the Ha-ras oncogene product p21. This suggests that the stereochemistry around the beta-phosphorus might be similar in these compounds.


Asunto(s)
Proteínas de Neoplasias/análisis , Nucleótidos/análisis , Oncogenes , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Magnesio/análisis , Espectroscopía de Resonancia Magnética , Proteína Oncogénica p21(ras) , Fósforo , Unión Proteica
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