RESUMO
As a consequence of environmental protection and legal restrictions, increasing efforts are made to avoid radioactivity. One alternative is the labelling of ligands with chemiluminescent acridinium esters such as 2,6-dimethyl-4-(N-succinimidyloxy-carbonyl)phenyl 10-methylacridinium-9-carboxylate methosulphate (DMAE-NHS). When exposed to hydrogen peroxide in a basic solution, the DMAE-moiety decays with emission of a short-lasting chemiluminescent flash. With the goal of replacing the radioactive label in protein ligands with a DMAE label, and of increasing the efficiency by using microtitre plate technology for DMAE detection, we compared the receptor binding properties of iodinated interleukin-1 alpha (125I-IL-1 alpha), interleukin-1 beta (125I-IL-1 beta) and interferon-gamma (125I-IFN-gamma) with the corresponding DMAE-labelled ligands. The luminescence signal was assessed in a single-tube luminometer and in the prototype of a chemiluminescent microtitre plate reader. Derivatization of the three proteins with DMAE-N-hydroxy-succinimide resulted in photon yields of up to 100,000 counts per femtomole. As shown by Scatchard analysis, no significant loss of receptor binding affinity was observed, which might have been expected as a consequence of the chemical modification of the proteins. The use of DMAE labelling of proteins has the following advantages as compared to iodination: (i) the coupling reaction and binding assay can be performed in a normal laboratory, (ii) since there is no radiolysis, the DMAE-labelled proteins remain stable, (iii) the detection sensitivity may be improved as a consequence of higher specific activity of the DMAE label.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Acridinas/metabolismo , Interferon gama/metabolismo , Receptores de Interferon/análise , Receptores de Interleucina-1/análise , Humanos , Peróxido de Hidrogênio , Radioisótopos do Iodo , Cinética , Medições Luminescentes , Receptores de Interferon/metabolismo , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/metabolismo , Receptor de Interferon gamaRESUMO
Interleukin-1 (IL-1) alpha and beta are polypeptide hormones that mediate a broad range of biological activities and interact with surface receptors on numerous cell types. Great efforts are made at present to define the interaction domain of IL-1 with its receptor. We have tried to map the domain of IL-1 beta by assessing the receptor interaction of synthetic octapeptide acid amides representing overlapping segments of the IL-1 beta primary sequence. Since the tertiary structure of IL-1 beta is known, the selection of octapeptides could be confined to the surface exposed residues. More than a 100 octapeptides were tested for binding in a competitive binding assay, using a mouse thymoma cell line (EL 4.61) as a receptor source and 125I-IL-1 alpha and beta as radioligands. No binding was found at up to a one hundred fold excess of octapeptide over radioligand. From this lack of binding we conclude that the entropic cost of conformationally freezing the octapeptide is high and that the conformation of the binding domain is per se in terms of free energy and is stabilized by the overall tertiary structure of IL-1.
Assuntos
Interleucina-1/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptores Imunológicos/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Receptores de Interleucina-1 , Células Tumorais CultivadasRESUMO
A position-sensitive single-photon counting imaging system, which can determine intensity and location of a light source, has been used for the detection of 125I-labeled Interleukin-1 and [3H]thymidine in 96 wells of a microtiter plate (MTP) simultaneously. 4 Bq (1 Bq = 1 Bequerel = 1 disintegration/s), 22 and 150 Bq of 125I and 3, 10 and 100 Bq of 3H were visualized and quantified by transforming the radioactivity into light in the visible range by means of Xtalscint, a solid scintillator. After only 1 min of photon accumulation time, the highest radioactivity of both isotopes could be clearly distinguished from background. Photon counts correlate well with radioactivity measured in a beta-counter (for 3H) and a gamma-counter (for 125I). The overall counting efficiency was about 5% for 125I and 3% for 3H.
Assuntos
Radiometria/métodos , Animais , Linhagem Celular , Estudos de Avaliação como Assunto , Radioisótopos do Iodo/análise , Radiometria/instrumentação , Contagem de Cintilação , Timidina/análiseAssuntos
Antibióticos Antineoplásicos , Neoplasias Mamárias Experimentais/prevenção & controle , Rifamicinas/farmacologia , Tecido Adiposo/metabolismo , Administração Oral , Animais , Benzo(a)Antracenos , Compostos de Benzil/administração & dosagem , Compostos de Benzil/metabolismo , Compostos de Benzil/farmacologia , Feminino , Injeções Intraperitoneais , Mucosa Intestinal/metabolismo , Rim/metabolismo , Fígado/metabolismo , Músculos/metabolismo , Neoplasias Experimentais/induzido quimicamente , Ratos , Rifampina/sangue , Rifamicinas/administração & dosagem , Rifamicinas/metabolismo , Baço/metabolismo , TrítioRESUMO
One of the most potent inhibitors of RNA-dependent DNA polymerase activity so far described (rifazacyclo-16) was not correspondingly as active in focus inhibition. This discrepancy was thought to be due to the inability of the drug to penetrate the cell membrane. It has been found that a very low level of amphotericin B allows this drug, as well as the previously described 2',6'-dimethyl-N(4')benzyl-N(4')-[desmethyl] rifampicin, to exhibit a very high capability to inhibit focus formation. Since these two drugs are highly lipophilie, their activity may be expected to be dependent upon any lipophilic components in the medium, such as serum or detergents. The use of amphotericin B as well as serum in tissue cultures is common, and could account for some of the variability in focus inhibition reported in the literature.
Assuntos
Anfotericina B/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Rifamicinas/farmacologia , Animais , Linhagem Celular , Sinergismo Farmacológico , Concentração de Íons de Hidrogênio , Camundongos , Vírus da Leucemia Murina de Moloney/crescimento & desenvolvimento , Ensaio de Placa ViralRESUMO
A reverse transcriptase activity, extracted from virus-transformed cells, is activated by very low concentrations of nonionic detergents. These same detergents also significantly reduce the effectiveness of certain rifamycin derivatives as inhibitors of the polymerase activity when the detergents are present at micelle-forming concentrations.
Assuntos
Detergentes/farmacologia , DNA Polimerase Dirigida por RNA , Rifamicinas/farmacologia , Transformação Celular Neoplásica , Células Cultivadas , Ativação Enzimática , Inibidores da Transcriptase Reversa , Rifamicinas/antagonistas & inibidoresRESUMO
It is shown that rifampicin, and especially the related antibiotic 2',6'-dimethyl-N(4')-benzyl-N(4')- [desmethyl]rifampicin (DMB-rifampicin) can inhibit focus formation by Moloney sarcoma virus on BALB/3T3 tissue cultures. At 10 mug/ml DMB-rifampicin totally inhibits focus formation while reducing virus replication by at least a factor of fifty and cell proliferation by only a factor of three. These observations, taken together with those of others, suggest a role for an RNA-dependent DNA polymerase and the gene for its synthesis both in normal cell processes and in the transformation process.