RESUMO
PURPOSE: To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). METHODS: Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. RESULTS: Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. CONCLUSIONS: Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].
Assuntos
Substância Própria/ultraestrutura , Cirurgia da Córnea a Laser , Ceratite/etiologia , Ceratomileuse Assistida por Excimer Laser In Situ , Lasers de Excimer/uso terapêutico , Cicatrização/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Antígeno CD11b/metabolismo , Substância Própria/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Digoxigenina/análogos & derivados , Digoxigenina/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Ceratite/metabolismo , Antígeno Ki-67/metabolismo , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Técnicas de Cultura de Órgãos , Doadores de TecidosRESUMO
We have synthesized a novel derivative of Digitoxin, termed "MonoD", which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within 1 h of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles, and increased expression of pro-autophagic markers Beclin-1 and LC3-II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3-II and Beclin-1. Prolonged treatment with MonoD-induced apoptosis in lung cancer cells. We investigated MonoD-dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3-II and Beclin-1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD-dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. J. Cell. Physiol. 231: 817-828, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Digitoxina/análogos & derivados , Digitoxina/farmacologia , Neoplasias Pulmonares/patologia , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Digoxigenina/análogos & derivados , Digoxigenina/farmacologia , Humanos , Neoplasias Pulmonares/metabolismo , Modelos Biológicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Superóxidos/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-ß-D-glucoside.
Assuntos
Digoxigenina/análogos & derivados , Digoxigenina/química , Glucosídeos/síntese química , Glicosiltransferases/metabolismo , Esteroides/química , Catálise , Digoxigenina/síntese química , Digoxigenina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Glucosídeos/química , Glicosilação , Glicosiltransferases/química , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , EstereoisomerismoRESUMO
We describe a new family of uronium-type coupling reagents that differ in their iminium moieties and leaving groups. The presence of the morpholino group in conjunction with an oxime derivative--especially ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma)--had a marked influence on the solubilities, stabilities, and reactivities of the reagents. Finally, the new uronium salt derived from Oxyma (COMU) performed extremely well in the presence of only 1 equiv of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. COMU also showed a less hazardous safety profile than the benzotriazole-based HDMA and HDMB, which exhibited unpredictable autocatalytic decompositions. Furthermore, the Oxyma moiety contained in COMU suggests a lower risk of explosion than in the case of the benzotriazole derivatives.
Assuntos
Morfolinas/química , Compostos Organofosforados/química , Peptídeos/síntese química , Triazóis/química , Compostos Aza/química , Cristalografia por Raios X , Digoxigenina/análogos & derivados , Digoxigenina/química , Conformação Molecular , Peptídeos/química , Pregnadienos/química , Succinimidas/químicaRESUMO
We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG antibody. The whole procedure can be done in about a day and has the following advantages: It is highly sensitive, specificity is confirmed by monitoring the size of the RT-PCR product, it is non-radioactive, quantitative, and does not require expensive specialized equipment.
Assuntos
Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Southern Blotting , Primers do DNA , DNA Complementar , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Eletroforese em Gel de Ágar , Humanos , Medições Luminescentes , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Padrões de Referência , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
The development of 24-colour fluorescence in situ hybridisation (FISH) has led to significant advances in cytogenetic research and offers the potential for automated karyotypic analysis. However, these techniques are not in routine research or clinical use because of limitations in methods of probe preparation. This article presents new probe construction protocols and strategies for multiple-colour karyotyping by chromosome painting, which makes the technique more efficient and may lead to more widespread implementation. We used paints generated by our protocols to demonstrate the presence of a cryptic translocation t(13;11;22) in the paediatric sarcoma cell line RMS 1598.
Assuntos
Coloração Cromossômica/métodos , Hibridização in Situ Fluorescente/métodos , DNA de Neoplasias/análise , Nucleotídeos de Desoxiuracil/metabolismo , Digoxigenina/análogos & derivados , Digoxigenina/metabolismo , Humanos , Cariotipagem/métodos , Reação em Cadeia da Polimerase/métodos , Sarcoma/química , Células Tumorais CultivadasRESUMO
BACKGROUND: The balance among cell proliferation, cell differentiation, and cell death determines the cell number in a population as well as the size or even the stage of a tumor. Thus, to improve our understanding of the pathogenesis of neoplasms, it is important to investigate the regulation of both cell proliferation and cell death. METHODS: This study examined the occurrence of apoptosis and proliferative capacity in 46 breast carcinomas: 20 intraductal carcinomas (ductal carcinomas in situ [DCIS]) and 26 infiltrative ductal carcinomas (IDC). Terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) and immunostaining with the Ki-67 antibody were used in the examination. A ladder of DNA fragments induced by apoptosis was demonstrated by means of DNA agarose gel electrophoresis in 10 of the available TUNEL positive and negative samples. RESULTS: The results were correlated with p53, bcl-2, estrogen receptor (ER), and progesterone receptor (PR) protein expression, which would suggest association with apoptosis by immunohistochemistry. The apoptosis and proliferation of each cancer were expressed as the number of tumor cells undergoing apoptosis and proliferation per 1000 tumor cells. The extent of apoptosis was more frequently observed in DCIS than in IDC (21.9+/-6.8 vs. 4.0+/-0.9, P < 0.001), and the proliferation activity was significantly higher in IDC than in DCIS (16.8+/-6.5 vs. 3.5+/-0.8, P < 0.006). Apoptosis associated with MIB-1 positive cells and TUNEL labeling was significantly higher in IDC than in DCIS (3.26 vs. 0.42, P=0.001). In DCIS, apoptosis was correlated with p53 (r=0.663, P=0.005), and p53 had a reverse correlation with bcl-2 (r=0.620, P= 0.018). Moreover, bcl-2 expression was associated with ER (P=0.028) and PR (P= 0.005) expression in both DCIS and IDC. CONCLUSIONS: The results of this study show that a higher degree of apoptosis and lower proliferation activity in intraductal carcinoma result in a steady-state, self-renewing condition in which net growth of the tumor is rare. The results also indicate that apoptosis was altered by the expression of p53, bcl-2, ER, and PR.
Assuntos
Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/patologia , Genes bcl-2/genética , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Mama/genética , Carcinoma in Situ/genética , Carcinoma Ductal de Mama/genética , Divisão Celular , Sobrevivência Celular , Fragmentação do DNA , DNA Nucleotidilexotransferase/fisiologia , DNA de Neoplasias/análise , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Feminino , Humanos , Imuno-Histoquímica , Antígeno Ki-67 , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteína Supressora de Tumor p53/genéticaRESUMO
A one-step procedure for the synthesis of different tyramide conjugates, which can be utilized in the catalyzed reporter deposition (CARD) amplification system, is described. Succinimidyl esters of biotin, digoxigenin, and of the fluorochromes fluorescein, rhodamine, aminomethylcoumarine acetic acid, and Cy3 were coupled to tyramine in dimethylformamide (DMF) adjusted to a pH of 7.0-8.0 with triethylamine (TEA). The coupling reaction can be performed within 2 hr and the reaction mixture can be applied without further purification steps. Furthermore, trinitrophenyl (TNP)-tyramide was prepared by adding 2,4,6,-trinitrobenzenesulfonic acid to tyramine dissolved in either MilliQ/DMF basified with TEA or in an NaHCO3 (pH 9.5) buffer. A subsequent precipitation of the TNP-tyramide resulted in a high-yield isolation of this conjugate. The synthesized tyramide conjugates were applied successfully in single- and multiple-target in situ hybridization (ISH) procedures to detect both repetitive and single-copy DNA target sequences in cell preparations with high efficiency. The described approach provides an easy and fast method to prepare a variety of tyramide conjugates in bulk amounts at relatively low cost.
Assuntos
Biotina/síntese química , Digoxigenina/síntese química , Corantes Fluorescentes/síntese química , Hibridização In Situ/métodos , Trinitrobenzenos/síntese química , Tiramina/análogos & derivados , Tiramina/síntese química , Biotina/análogos & derivados , Carcinoma de Células de Transição/genética , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 12/genética , Digoxigenina/análogos & derivados , Humanos , Sensibilidade e Especificidade , Células Tumorais CultivadasRESUMO
Searching for a binding protein in blood, which may be involved in the specific transport of cardiac glycosides to their receptor sites on the sodium pump, we isolated a cardiac glycoside-binding protein (CGBG) of 26 kDa from the globulin fraction of bovine serum by affinity chromatography and on a ouabain-Sepharose 4B column by a purification factor of 5000. The cardiac glycoside-binding globulin was labeled specifically and covalently by the protein-reactive digoxigenin derivative HDMA (N-hydroxysuccimidyldigoxigenin-3-O-methylcarbonyl-epsilon-+ ++aminocapro ate). Even very high concentrations of other steroids, such as estrogen, testosterone, progesterone, and cortisone, did not prevent HDMA-labeling (at 5 and 100 nM) of CGBG, but the cardenolides ouabain and digoxin or the bufadienolide proscillaridin A did so. CGBG is a homodimer of two 26-kDa subunits forming disulfide bonds, since HDMA labeling of a protein of 53 kDa was observed in SDS-polyacrylamide gel electrophoresis when beta-mercaptoethanol was absent during SDS denaturation. The N-terminal amino acid sequence K-D-V-Y-R-A-P-D-G-T-Q-S-A showed no sequence similarity with proteins recorded in gene and protein sequence data banks. A 90-kDa cytosolic CGBG exists in bovine kidneys and reacts with antibodies against CGBG. Binding of ouabain to the cardiac glycoside-binding globulin was monitored by quenching of intrinsic tryptophan fluorescence. Such studies reveal two negatively cooperative ouabain binding sites with Kd' of 1.52 nM and Kd' = 75 nM and with an interaction factor of 50 using a Koshland-Némethy-Filmer model. The demonstration of a cardiac glycoside-binding globulin in plasma is consistent with the recent finding of endogenous cardiac glycosides in mammals.
Assuntos
Glicosídeos Cardíacos/sangue , Soroglobulinas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia de Afinidade , Digoxigenina/análogos & derivados , Digoxigenina/metabolismo , Cinética , Mercaptoetanol/farmacologia , Dados de Sequência Molecular , Peso Molecular , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/imunologia , Espectrometria de Fluorescência , Succinimidas/metabolismoRESUMO
1. Which angiogenic growth factors actually mediate tumor growth in ethylnitrosourea (ENU)-induced gliomas in rats was examined. 2. In situ hybridization histochemistry with digoxigenin-labeled oligonucleotide probes was used to investigate the cellular expression and distribution of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNAs in ENU-induced gliomas. 3. Both VEGF and bFGF mRNAs were not detected in normal gial cells but in ENU-induced glioma cells. 4. Our results suggest that the growth of ENU-induced glioma may be regulated by multiple angiogenic growth factors and that these gliomas may proliferate by synthesizing such growth factors.
Assuntos
Fatores de Crescimento Endotelial/genética , Fator 2 de Crescimento de Fibroblastos/genética , Glioma/induzido quimicamente , Glioma/genética , Linfocinas/genética , RNA Mensageiro/metabolismo , Animais , Sequência de Bases , Digoxigenina/análogos & derivados , Etilnitrosoureia , Feminino , Glioma/patologia , Hibridização In Situ , Masculino , Dados de Sequência Molecular , Neoplasias Experimentais , Neuroglia , Sondas de Oligonucleotídeos , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
A flow cytometric method has recently been developed using biotinylated dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) to identify the endonuclease-induced DNA strand breaks occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the present study, we compared this method with one using digoxigenin-conjugated dUTP (d-dUTP) to label apoptotic cells. The discrimination of apoptotic from nonapoptotic cells was similar when incorporation of d-dUTP was compared with b-dUTP. Both techniques resulted in a 20-30 fold increase in staining of apoptotic over nonapoptotic cells although somewhat less background fluorescence was observed with the d-dUTP. Direct labeling with fluoresceinated dUTP (f-dUTP) was less sensitive in detecting DNA strand breaks, but had the advantage of simplicity. The principle of labeling DNA strand breaks using TdT was also employed to identify DNA replicating cells. To this end, the cells were incubated in the presence of BrdUrd, then exposed to UV light to selectively photolyse DNA containing the incorporated BrdUrd. DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This approach is an alternative to immunocytochemical detection of BrdUrd incorporation, but unlike the latter does not require prior DNA denaturation, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replication of less than 1% of the cell's genome. The discrimination between apoptotic vs. BrdUrd incorporating-cells is based on different extractability of DNA following cell fixation. This method can be applied to analyze both cell proliferation (DNA replication) and death (by apoptosis) in a single measurement.
Assuntos
Apoptose/fisiologia , Biotina/análogos & derivados , DNA/metabolismo , Nucleotídeos de Desoxiuracil/metabolismo , Digoxigenina/análogos & derivados , Fluoresceínas/metabolismo , Biotina/metabolismo , Medula Óssea/patologia , Bromodesoxiuridina/metabolismo , Camptotecina/farmacologia , Divisão Celular/fisiologia , DNA/efeitos dos fármacos , DNA Nucleotidilexotransferase/metabolismo , Sondas de DNA/metabolismo , Digoxigenina/metabolismo , Citometria de Fluxo/métodos , Células HL-60/química , Células HL-60/efeitos dos fármacos , Células HL-60/patologia , Humanos , Imuno-Histoquímica , Indicadores e Reagentes/metabolismo , Kit de Reagentes para DiagnósticoRESUMO
The digoxigenin derivative N-hydroxysuccinimidyl digoxigenin-3-O-methylcarbonyl-epsilon-aminocaproate (HDMA) has been shown to covalently label the ouabain binding site of the Na,K-ATPase epsilon subunit [Antolovic et al. (1995) Eur. J. Biochem. 227, 61-67]. In the present study we observed both, labeling and inactivation of the activity, of wild type Na,K-ATPase overexpressed in Xenopus oocyte. In contrast, no significant inhibition and no labeling could be detected when a Cys-113 of the first transmembrane segment was mutated to serine, although the affinity of this mutant for digoxigenin or HDMA measured in acute inhibition experiments was similar to the wild type. This indicates that after docking of its genin moiety, HDMA can form a thioester bond with Cys-113.
Assuntos
Marcadores de Afinidade , Cisteína/análise , Digoxigenina/análogos & derivados , Succinimidas , Animais , Mutação , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , XenopusRESUMO
Na+/K(+)-ATPase from pig kidney is inactivated by protein-reactive N-hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein-reactive derivatives N-hydroxysuccinimidyl digoxigenin-3-methylcarbonyl-epsilon-aminocaproate (HDMA), 3-amino-3-deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3-iodoacetylamino-3-deoxydigoxigenin (IAD) and digoxigenin-3-O-succinyl-[2-(N-maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37 degrees C, half-maximal inhibition of Na+/K(+)-ATPase was seen by HDMA at 0.47 microM, by ADHS at 5.8 microM, by IAD at 8 microM and by DSME at 94 microM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K(+)-ATPase. Affinity labeling of the alpha subunit by 'front door' or 'back door' phosphorylation was only seen with HDMA or ADHS in the range 0.1 microM. Excess of ouabain protected against affinity labeling. All the other protein-reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain. Tryptic hydrolysis of the HDMA-modified Na+/K(+)-ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2-kDa and 12.5-kDa peptides started amino-terminally with Asp68, and the 20-kDa peptide with Asp24. Thus, the HDMA-labeled peptides originate from the cardioactive steroid-binding site formed by the first and second transmembrane helix. N-Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside-binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cys104 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2-14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site-directed mutagenesis experiment [Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) EMBO J. 11, 1681-1687], that a cysteine residue (probably Cys104) participates in the structure and function of the cardiac glycoside binding.
Assuntos
Glicosídeos Cardíacos/metabolismo , Digoxigenina/análogos & derivados , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Succinimidas/química , Compostos de Sulfidrila/química , Marcadores de Afinidade , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Simulação por Computador , Digoxigenina/química , Hidrólise , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Tripsina/químicaRESUMO
Archival pathological specimens are a source of RNA and DNA for clinical surveillance or retrospective studies. We employed a modification of the acid guanidium thiocyanate-phenol-chloroform extraction method for the recovery of total RNA from formalin-fixed, paraffin-embedded neoplastic thyroid tissue. The extracted RNA was used for reverse transcription of ptc and subsequent amplification of the complementary DNA (cDNA) by the reverse transcription-polymerase chain reaction (RT-PCR). In lieu of 32P-labeled DNA for hybridization studies, we supplemented the nucleotide pool in the amplification reaction with a modified pyrimidine, digoxigenin-11-dUTP. Digoxigenin-11-dUTP was incorporated directly into the PCR product, eliminating the need for hybridization, posthybridization washes, and prolonged autoradiography. These products were resolved by electrophoresis on agarose gels, Southern blotted to nylon membranes, and rapidly detected by chemiluminescence. This nonradioisotopic method has expedited and reduced the cost for molecular investigations with archival pathological specimens by providing equal sensitivity to or greater sensitivity than that of DNA-labeled radionuclides without the associated biological hazards.
Assuntos
Carcinoma Papilar/genética , Reação em Cadeia da Polimerase/métodos , Proto-Oncogenes/genética , Neoplasias da Glândula Tireoide/genética , Primers do DNA , Nucleotídeos de Desoxiuracil/química , Digoxigenina/análogos & derivados , Digoxigenina/química , Eletroforese em Gel de Ágar/métodos , Formaldeído , Humanos , Indicadores e Reagentes , Hibridização de Ácido Nucleico , Inclusão em Parafina , Radioisótopos de Fósforo , RNA/isolamento & purificação , DNA Polimerase Dirigida por RNA , Glândula Tireoide/patologia , Fixação de Tecidos , Microglobulina beta-2RESUMO
Our objective was to identify commercially available digoxin immunoassays whose cross-reactivity with digoxin metabolites paralleled the pharmacological activity of the metabolites. We measured the immunoreactivity of digoxigenin bis- and monodigitoxosides, digoxigenin, and dihydrodigoxin in four immunoassays and compared the immunoactivities with pharmacological activities from studies involving whole-animal and receptor (Na,K-ATPase)-based assays. Correlation coefficients for comparisons of immunoassay reactivity and human heart receptor reactivities were: ACS, 0.96; TDx, 0.60; Stratus, 0.57; and Magic, 0.42. Comparison with other biological assays showed a similar trend. The major difference in metabolite cross-reactivities among the immunoassays was that of digoxigenin (ACS, 0.7%; TDx, 103%; Stratus, 108%; Magic, 153%), which has approximately 10% bioactivity relative to digoxin. Measured recovery of mixtures of digoxin and metabolites confirmed these findings. We conclude that the monoclonal antibody in the ACS digoxin assay closely mimics Na,K-ATPase in detecting digoxin and its metabolites. This finding provides a basis for developing therapeutic drug monitoring immunoassays capable of approximating the true pharmacological activity of a mixture of drug metabolites.
Assuntos
Digoxina/sangue , Imunoensaio/estatística & dados numéricos , Animais , Bioensaio , Gatos , Digoxigenina/análogos & derivados , Digoxigenina/sangue , Digoxigenina/farmacologia , Digoxina/análogos & derivados , Digoxina/farmacologia , Cobaias , Coração/efeitos dos fármacos , Humanos , Camundongos , Ouabaína/metabolismo , Sensibilidade e Especificidade , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
The liver plays a key regulatory role in cholesterol metabolism. Two proteins are central in this role; the LDL receptor and 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), the rate-limiting enzyme in cholesterol biosynthesis. In the current investigation, we have used a sensitive nonradioactive method to study the regulation of LDL receptor and HMG CoA reductase mRNA levels in liver biopsy samples and freshly isolated mononuclear leukocytes from 13 patients who underwent cholecystectomy for gallstones. mRNA copy numbers were determined by PCR amplification of reverse-transcribed RNA using synthetic RNA as an internal standard. Incorporation of digoxigenin-11-dUTP during amplification allowed direct detection and quantitation of mRNA levels by chemiluminescence. These experiments showed that the average number of LDL receptor mRNA molecules in liver (21 +/- 3 x 10(4)/micrograms of RNA) and mononuclear leukocytes (24 +/- 3 x 10(4)/micrograms of RNA) are indistinguishable, whereas the number of HMG CoA reductase molecules in liver (107 +/- 15 x 10(4)/micrograms of RNA) is smaller than that in mononuclear leukocytes (158 +/- 21 x 10(4)/micrograms of RNA, P < 0.05). These numbers correspond to an average of 1-6 copies of LDL receptor mRNA and 5-42 copies of HMG CoA reductase mRNA per cell. There was a significant correlation between the numbers of LDL receptor (P = 0.0005) and HMG CoA reductase (P = 0.003) mRNA molecules in liver and mononuclear leukocytes. Furthermore, the numbers of copies of HMG CoA reductase and LDL receptor mRNA were correlated with each other in both liver (P = 0.02) and mononuclear leukocytes (P = 0.01), consistent with coordinate regulation. These data demonstrate that the mechanisms which regulate mRNA levels in liver and mononuclear cells are similar and suggest that freshly isolated mononuclear cells can be used to predict HMG CoA reductase and LDL receptor mRNA levels in liver.
Assuntos
Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Redutases/biossíntese , Leucócitos Mononucleares/metabolismo , Fígado/metabolismo , Receptores de LDL/biossíntese , Adulto , Idoso , Sequência de Bases , Biópsia , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Feminino , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Modelos Lineares , Medições Luminescentes , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Receptores de LDL/genéticaRESUMO
An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.
Assuntos
HIV-1/enzimologia , Leucócitos Mononucleares/microbiologia , Poli A , DNA Polimerase Dirigida por RNA/metabolismo , Biotina , Colorimetria , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Ensaio de Imunoadsorção Enzimática , Transcriptase Reversa do HIV , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Indicadores e Reagentes , Retroviridae/enzimologia , Sensibilidade e EspecificidadeRESUMO
Tay-Sachs disease (TSD, GM2 gangliosidosis, Type I) is an autosomal recessive lysosomal storage disease caused by deficiency of beta-hexosaminidase A (Hex A) resulting from mutations in the gene (HEXA) encoding the alpha-subunit of the enzyme. Three mutations, in exons 7 and 11 and at the exon 12-intron 12 junction, account for > 90% of alleles identified in obligate Ashkenazi Jewish carriers. Mutation analysis requires amplification of available DNA by separate polymerase chain reactions (PCRs) and either restriction digestion and gel electrophoresis or 32P-labeled allele-specific oligonucleotide (ASO) probes. We developed a simple, nonradioisotopic method for rapidly identifying TSD carriers by a triplex PCR reaction followed by dot-blot analysis, using three wild-type and three mutant ASOs end-labeled with digoxigenin-dUTP (dig-ASO). Hybridization was demonstrated immunologically by reaction with an anti-digoxigenin-alkaline phosphatase conjugate followed by colorimetric demonstration of phosphatase activity. The results of analyses by the dig-ASO method of 65 carriers identified by serum enzyme activity and of 6 high-risk fetuses in prenatal testing were the same as those obtained by more conventional restriction analysis. Dig-ASO testing correctly reclassified 10 individuals who had tested inconclusively on analysis for leukocyte beta-hexosaminidase A activity; 3 were identified as carriers and 7 as noncarriers. The simplicity of the assay and the avoidance of the radioisotopes make this a potentially useful method for TSD carrier detection by mutation analysis in Ashkenazi Jews from populations in whom the identity and frequencies of the common TSD mutations are known.
Assuntos
Triagem de Portadores Genéticos/métodos , Testes Genéticos/métodos , Judeus , Doença de Tay-Sachs/genética , Colorimetria , DNA/genética , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Éxons , Feminino , Hexosaminidase A , Humanos , Técnicas Imunoenzimáticas , Indicadores e Reagentes , Íntrons , Mutação , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Gravidez , Diagnóstico Pré-Natal , Doença de Tay-Sachs/diagnóstico , beta-N-Acetil-Hexosaminidases/deficiência , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
A sensitive nonradioactive ribunuclease protection assay is described which we have used to study c-myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)-rUTP instead of [alpha-32P]CTP or [alpha-32P]UTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.
Assuntos
Genes myc , RNA Mensageiro/genética , Ribonucleases , Carcinoma de Células Pequenas/genética , Nucleotídeos de Desoxiuracil , Digoxigenina/análogos & derivados , Eletroforese em Gel de Poliacrilamida , Humanos , Indicadores e Reagentes , Medições Luminescentes , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas , RNA Mensageiro/análise , Transcrição Gênica , Células Tumorais Cultivadas/metabolismoRESUMO
This report describes the development of a method to measure mRNA in small samples of human tissue by the polymerase chain reaction with a nonradioactive label. In this method RNA is reverse-transcribed in the presence of a control RNA, and subsequently amplified by the polymerase chain reaction during which a nonradioactive label (digoxigenin-11-dUTP) is incorporated. Gel blotting and immunological detection of digoxigenin followed by a chemiluminescent reaction provide an intense signal on film. This allows the detection and quantitation of 3-hydroxy-3-methylglutaryl (HMG) CoA reductase mRNA in 12 ng of RNA. We demonstrate that this is a sensitive and reproducible method, and that quantitation is linear with respect to the amount of mRNA present. The application of this method to the measurement of low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA levels in circulating peripheral blood mononuclear cells and human liver biopsy samples is discussed. The use of chemiluminescent reagents instead of radioactive labels allows this procedure to be performed safely in laboratories not equipped for radioactivity.