RESUMEN
PURPOSE: The objective of this trial was to evaluate the safety and efficacy of melatonin oral gel mouthwashes in the prevention and treatment of oral mucositis (OM) in patients treated with concurrent radiation and systemic treatment for head and neck cancer. METHODS: Randomized, phase II, double-blind, placebo-controlled trial (1:1 ratio) of 3% melatonin oral gel mouthwashes vs. placebo, during IMRT (total dose ≥ 66 Gy) plus concurrent Q3W cisplatin or cetuximab. Primary endpoint: grade 3-4 OM or Severe Oral Mucositis (SOM) incidence by RTOG, NCI, and a composite RTOG-NCI scales. Secondary endpoints: SOM duration and grade 2-4 OM or Ulcerative Oral Mucositis (UOM) incidence and duration. RESULTS: Eighty-four patients were included in the study. Concurrent systemic treatments were cisplatin (n = 54; 64%) or cetuximab (n = 30; 36%). Compared with the placebo arm, RTOG-defined SOM incidence was numerically lower in the 3% melatonin oral gel arm (53 vs. 64%, P = 0.36). In patients treated with cisplatin, assessed by the RTOG-NCI composite scale, both SOM incidence (44 vs. 78%; P = 0.02) and median SOM duration (0 vs. 22 days; P = 0.022) were significantly reduced in the melatonin arm. Median UOM duration assessed by the RTOG-NCI scale was also significantly shorter in the melatonin arm (49 vs. 73 days; P = 0.014). Rate of adverse events and overall response rate were similar between the two arms. CONCLUSIONS: Treatment with melatonin oral gel showed a consistent trend to lower incidence and shorter SOM duration and shorter duration of UOM. These results warrant further investigation in phase III clinical trial.
Asunto(s)
Antineoplásicos/efectos adversos , Antioxidantes/administración & dosificación , Quimioradioterapia/efectos adversos , Melatonina/administración & dosificación , Antisépticos Bucales/administración & dosificación , Estomatitis/prevención & control , Administración Oral , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/administración & dosificación , Antioxidantes/efectos adversos , Cetuximab/administración & dosificación , Cetuximab/efectos adversos , Cisplatino/administración & dosificación , Cisplatino/efectos adversos , Método Doble Ciego , Femenino , Geles/administración & dosificación , Neoplasias de Cabeza y Cuello , Humanos , Incidencia , Masculino , Melatonina/efectos adversos , Persona de Mediana Edad , Antisépticos Bucales/efectos adversos , Placebos/administración & dosificación , Prueba de Estudio Conceptual , Estudios Prospectivos , Estomatitis/epidemiología , Estomatitis/etiologíaRESUMEN
The catalytic subunit of protein kinase CK2 (CK2alpha) was found associated with heterogeneous nuclear ribonucleoprotein particles (hnRNPs) that contain the core proteins A2 and C1-C2. High levels of CK2 activity were also detected in these complexes. Phosphopeptide patterns of hnRNP A2 phosphorylated in vivo and in vitro by protein kinase CK2 were similar, suggesting that this kinase can phosphorylate hnRNPA2 in vivo. Binding experiments using human recombinant hnRNP A2, free human recombinant CK2alpha or CK2beta subunits, reconstituted CK2 holoenzyme and purified native rat liver CK2 indicated that hnRNP A2 associated with both catalytic and regulatory CK2 subunits, and that the interaction was independent of the presence of RNA. However, the capability of hnRNP A2 to bind to CK2 holoenzyme was lower than its binding to the isolated subunits. These data indicate that the association of CK2alpha with CK2beta interferes with the subsequent binding of hnRNP A2. HnRNP A2 inhibited the autophosphorylation of CK2beta. This effect was stronger with reconstituted human recombinant CK2 than with purified native rat liver CK2.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Quinasa de la Caseína II , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Hígado/metabolismo , Fosforilación , ARN Polimerasa II/metabolismo , RatasRESUMEN
Human renin has been expressed in Sf9 and CHO cells using two different gene constructs. The first construct contained a foreign signal peptide fused directly to the sequence encoding mature renin, whereas the second construct harbors the sequence for preprorenin. Prorenin was produced in significantly higher amounts than the mature enzyme expressed without its propeptide in both expression systems. Both directly expressed mature renin and proteolytically derived active renin have been purified and cocrystallized with the renin inhibitor Ro 42-5892. The 3D structure has been solved for both versions and demonstrates identity despite different glycosylation and different N termini.
Asunto(s)
Renina/química , Renina/genética , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Unión Competitiva , Western Blotting , Células CHO , Línea Celular , Cromatografía de Afinidad , Cricetinae , Cristalización , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Expresión Génica , Humanos , Imidazoles/farmacología , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Conformación Proteica , Radioinmunoensayo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Renina/aislamiento & purificación , Renina/metabolismo , SpodopteraRESUMEN
Protein kinase CK2 has been found in two nuclear fractions obtained after treatment of purified rat liver nuclei with nucleases (S1 fraction) and subsequently with 1.6 M NaCl (S2 fraction). In both fractions three isoforms of the alpha subunit were identified. Two of them corresponded to the classical alpha and alpha' subunits, whereas the identity of the third one (alpha 3) remains unknown. In the S1 fraction two peaks of CK2 activity were detected at 6 h (5.5 fold) and 24 h (1.9 fold) after partial hepatectomy, whereas no significant changes were found in the S2 fraction. At 6 h after laparatomy a much lower increase of CK2 in S1 fraction was also detected (2.5 fold). The increases in CK2 activity found at 6 h after hepatectomy or laparatomy were accompanied with rises in the amount of the alpha subunit.
Asunto(s)
Núcleo Celular/enzimología , Regeneración Hepática/fisiología , Hígado/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Quinasa de la Caseína II , Fraccionamiento Celular , Núcleo Celular/ultraestructura , Hepatectomía , Cinética , Masculino , Fosforilación , Proteínas Serina-Treonina Quinasas/aislamiento & purificación , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Heterogeneous nuclear ribonucleoproteins bind to RNA as long as it is transcribed. Since their binding can be sequence-specific, it has been suggested that their expression in different tissues could vary depending on the specific mRNA processing requirements. In order to better establish this possibility we studied the presence of the heterogeneous nuclear ribonucleoproteins A1, A2/B1, C and D in the cell nuclei of different rat tissues by one- and two-dimensional immunoblotting. We found that these proteins were heterogeneously distributed among tissues and that they were found in different proportions.
Asunto(s)
Núcleo Celular/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Ribonucleoproteínas/metabolismo , Empalme Alternativo , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Células HeLa , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Distribución TisularRESUMEN
It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei.
Asunto(s)
Calmodulina/farmacología , Núcleo Celular/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteínas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Quinasa de la Caseína II , Cromatografía de Afinidad , Guanosina Trifosfato/metabolismo , Células HeLa , Ribonucleoproteínas Nucleares Heterogéneas , Humanos , Masculino , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Ribonucleoproteínas/análisis , Ribonucleoproteínas/aislamiento & purificación , Homología de Secuencia de Aminoácido , Serina Endopeptidasas , Especificidad por SustratoRESUMEN
Casein kinase 2 was released from rat liver cells nuclei by digestion with DNase I plus RNase A. This treatment also released three major substrates of 50, 40-42, and 37 kDa. Casein kinase 2 and substrates were also extracted by DNase or RNase separately. However, in DNase extracts only the 37 kDa protein was phosphorylated by casein kinase 2, whereas in RNase extracts all three substrates were phosphorylated. When the DNase extracts were subsequently treated with RNase the 40-42 substrates were then phosphorylated, indicating that their interaction with RNA prevents their phosphorylation by casein kinase 2. The ratio of B: alpha subunits of casein kinase 2 present in the nuclease extracts was higher than that of the purified enzyme, which is assumed to be 1:1. A further analysis by sucrose gradient centrifugation revealed that under physiological salt conditions casein kinase 2 from nuclease extracts formed large aggregates (higher than 300 kDa) which were disrupted at 400 mM KCl. At the latter KCl concentration CK-2 activity was localized at a position corresponding to a M(r) of 230-250 kDa, which is still higher than the typical tetrameric form of the enzyme.
Asunto(s)
Núcleo Celular/enzimología , Desoxirribonucleasa I/metabolismo , Hígado/ultraestructura , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Ribonucleasa Pancreática/metabolismo , Animales , Caseína Quinasas , Centrifugación por Gradiente de Densidad , Peso Molecular , Fosfoproteínas/metabolismo , Fosforilación , Cloruro de Potasio , RatasRESUMEN
This report describes the immunological identification of a 60-kDa calmodulin-binding protein, previously detected in the nuclei of rat liver cells (Bachs, O., Lanini, L., Serratosa, J., Coll, M.J., Bastos, R., Aligué, R., Rius, E., and Carafoli, E. (1990) J. Biol. Chem. 265, 18595-18600), as the calmodulin-dependent protein phosphatase calcineurin. Calcineurin could be extracted from the nuclei by incubation with DNase and RNase, indicating that it is associated with nuclear structures sensitive to the action of nucleases (chromatin or/and ribonucleoproteins). The presence of calcineurin in the nuclei of rat liver cells indicates that calmodulin may modulate the phosphorylation level of nuclear proteins by promoting their dephosphorylation. This report also shows that calmodulin inhibits the activity of casein kinase-2 in the nuclear fractions obtained by nuclease extraction. Phosphorylation experiments indicate that casein kinase-2 phosphorylates three major substrates of 100, 42-44, and 37 kDa as well as other minor proteins in the nuclease extracts. Calmodulin reduces the phosphorylation level of the two latter major proteins and of a minor band of 50 kDa. Thus, nuclear calmodulin in rat liver cells could regulate phosphorylation of nuclear proteins by at least two mechanisms: 1) activation of calcineurin and 2) inhibition of casein kinase-2.
Asunto(s)
Calmodulina/metabolismo , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Animales , Western Blotting , Calcineurina , Proteínas de Unión a Calmodulina/metabolismo , Caseína Quinasas , Bovinos , Núcleo Celular/metabolismo , Electroforesis en Gel Bidimensional , Radioisótopos de Yodo , Masculino , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
By using a 125I-calmodulin overlay assay, three major high-affinity calmodulin-binding proteins, showing apparent molecular masses of 135, 60, and 50 kDa, have been detected in purified nuclear fractions isolated from rat neurons. It has been shown that after extraction of the nuclei with nucleases and high salt, all these proteins remain strongly associated with the nuclear matrix. The 60- and 50-kDa proteins have been previously identified as subunits of the calmodulin-dependent protein kinase II. We report here the immunoblot identification of the 135-kDa calmodulin-binding protein as myosin light chain kinase. We also show that the calmodulin-dependent protein phosphatase calcineurin is present in the neuronal nuclei and associated with the nuclear matrix. The nuclear localization of both calcineurin and myosin light chain kinase has been confirmed by immunocytochemical studies.