RESUMEN
BACKGROUND: TB control remains a serious public health problem, compounded by poor treatment adherence, which increases the likelihood of onward transmission. We evaluated the effectiveness of medication event reminder monitoring (MERM) upon treatment adherence in a high TB burden setting.METHODS: We conducted an open-label parallel group randomised controlled trial among pulmonary TB adults. Participants were provided with a MERM device to store their medications. In the intervention arm, the devices were set to provide daily medication intake reminders. Primary outcome was the proportion of patient-months in which at least 6/30 doses were missed. Secondary outcomes included 1) the proportion of patient-months in which at least 14/30 doses were missed, and 2) the proportion of doses missed.RESULTS: Of 2,142 patients screened, 798 (37.3%) met the inclusion criteria and 250 participants were enrolled. The mean ratio (MR) for poor adherence between the intervention and control groups was 0.72 (95% CI 0.55-0.86). The intervention was also associated with a reduction in the proportion of patients missing at least 14/30 doses (MR 0.61, 95% CI 0.54-0.68) and the percentage of total doses missed (MR 0.75, 95% CI 0.68-0.80).CONCLUSION: MERM is effective in improving TB treatment adherence in a resource-limited environment.
Asunto(s)
Cumplimiento de la Medicación , Tuberculosis Pulmonar , Adulto , Humanos , Sistemas Recordatorios , Tuberculosis Pulmonar/tratamiento farmacológico , Monitoreo de DrogasRESUMEN
The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Proteínas Proto-Oncogénicas c-mos/fisiología , Serina/fisiología , Células 3T3 , Animales , Células COS , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cromatografía , Electroforesis en Gel de Campo Pulsado , Regulación Enzimológica de la Expresión Génica , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Proteína Quinasa 3 Activada por Mitógenos , Mutagénesis Sitio-Dirigida , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-mos/metabolismo , Factores de Tiempo , Transfección , Ensayo de Tumor de Célula MadreRESUMEN
c-Mos is a germ cell-specific MAP kinase kinase kinase (MAPKKK) that plays an essential role during meiotic divisions of oocytes. c-Mos is a key component of an activity, cytostatic factor, required for metaphase II arrest of unfertilized eggs in vertebrates. To understand the regulation of c-Mos, we are investigating c-Mos-interacting proteins. We provide evidence that mouse c-Mos binds to Hsp70, a molecular chaperone. Hsp70 was found to associate with Mos ectopically expressed in COS-1 cells. Mos-Hsp70 complexes could be immunoprecipitated with both Mos and Hsp70 antibodies. Despite a low-abundance of Mos, the Hsp70 antibody immunoprecipitated Mos as the major protein. Of importance, the Mos protein present in anti-Hsp70 immunoprecipitates functioned as an active MAPKKK indicating that it is not grossly misfolded. It is known that c-Mos protein kinase activity in cell extracts of transfected COS-1 or NIH3T3 cells is labile. We found that the inclusion of adenosine triphosphate (ATP) in cell extracts protected against the loss of Mos kinase activity. In the absence of ATP from cell extracts, protein kinase activity of Mos was lost within 6 h on ice even though the Mos protein was not degraded and remained bound to Hsp70. Based on our identification of c-Mos-Hsp70 interaction, one of the roles of ATP may be to assist the regulation of c-Mos via ATP involvement in the protein-folding function of Hsp70 and possibly other molecular chaperones. We also detected by coimmunoprecipitation a physical association between endogenous c-Mos and Hsp70 in Xenopus eggs. To provide further evidence for the functional significance of Hsp70 interaction to Mos function, we show that the residue serine 3 in Mos, which is important for the regulation of protein kinase activity of Mos is also important for Hsp70 association.
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Proteínas HSP70 de Choque Térmico/fisiología , Proteínas Proto-Oncogénicas c-mos/fisiología , Animales , Células COS , RatonesRESUMEN
Tumor blood volume (BV), subject to both morphologic and physiologic influences, can be measured using contrast-enhanced magnetic resonance imaging (MRI). The aims of this study were to determine whether MRI enhanced with a macromolecular contrast medium (MMCM) could resolve differences in BV between different tumor types, between different regions within tumors, and within the same tumor in life and after death. Tumor BV estimates were based on the MRI signal intensity responses in the tumors and in reference venous blood following enhancement with a blood pool MMCM using two mammary adenocarcinoma models. Estimates of BV were made before and immediately following death. An in vitro measurement of tumor gadolinium concentration following death was correlated with MRI enhancement. Statistically significant differences (P < 0.05) were observed in MRI-estimated tumor BV between tumor subtypes, between in vivo and postmortem measurements, and between the tumor periphery and tumor centers. MRI assays enhanced with a macromolecular contrast agent can resolve blood volume differences between tumor types, between regions within the same tumor, and between vital and postmortem states.
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Artefactos , Imagen por Resonancia Magnética/métodos , Neoplasias Mamarias Experimentales/irrigación sanguínea , Albúminas , Animales , Medios de Contraste , Femenino , Gadolinio DTPA , Procesamiento de Imagen Asistido por Computador , Neoplasias Mamarias Experimentales/patología , Trasplante de Neoplasias , Ratas , Ratas Endogámicas F344RESUMEN
Mos is a germ cell-specific serine/threonine protein kinase that plays an important role during meiotic divisions of oocytes. Upon expression in somatic cells, Mos causes cell cycle perturbations leading to neoplastic transformation. Mos activates the MAP kinase pathway in both oocytes and transformed somatic cells. To determine the mechanism of cell cycle perturbation in mos-transformed cells, we examined the status of some key regulators of G1 phase. We provide evidence that Mos causes an elevation in the level of cyclin D1 in NIH/3T3 cells. As expected from the increased cyclin D1 level, mos transformation of NIH/3T3 cells caused an increase in the protein kinase activities of cyclin D1-Cdk4 and cyclin E-Cdk2 and induced hyperphosphorylation of the retinoblastoma protein. Of importance, the level of cyclin D1 was also elevated in eye lens of the c-mos-transgenic mice compared to normal mice. Our results indicate that the mechanism of cellular transformation by Mos involves an elevation in the level of cyclin D1 in somatic cells.
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Quinasas CDC2-CDC28 , Transformación Celular Neoplásica , Ciclina D1/biosíntesis , Genes mos , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Células 3T3 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quinasa 2 Dependiente de la Ciclina , Quinasa 4 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/metabolismo , Cristalino/metabolismo , Meiosis , Ratones , Ratones Transgénicos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-mos/biosíntesis , Transducción de SeñalRESUMEN
Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.
Asunto(s)
Proteínas Oncogénicas v-mos/metabolismo , Proteínas Proto-Oncogénicas c-mos/metabolismo , Serina/metabolismo , Células 3T3 , Animales , Células COS , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Oncogénicas v-mos/genética , Fosforilación , Proteínas Proto-Oncogénicas c-mos/genética , Conejos , Relación Estructura-Actividad , XenopusRESUMEN
Macromolecular contrast medium-enhanced magnetic resonance imaging (MRI) and tumor-volume measurements were applied to monitor the effects of anti-vascular endothelial growth factor (anti-VEGF) antibody on microvascular characteristics and tumor growth of MDA-MB-435 human breast cancer cells implanted in nude rats. Administration of anti-VEGF antibody (three 1 mg doses at 3-day intervals) induced significant reductions in tumor growth rates (p < 0.05) and in MRI-assayed microvascular permeabilities (p < 0.05). Results of the study were consistent with previous observations that new microvessels formed in response to angiogenesis are hyperpermeable, and with the hypothesis that hyperpermeability is a mechanistic element in angiogenesis. Variations in tumor-vessel hyperpermeability can be measured by contrast-enhanced MRI, which may prove useful for assessing antiangiogenesis therapy.
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Adenocarcinoma/irrigación sanguínea , Anticuerpos/farmacología , Neoplasias de la Mama/irrigación sanguínea , Permeabilidad Capilar/efectos de los fármacos , Factores de Crecimiento Endotelial/inmunología , Linfocinas/inmunología , Adenocarcinoma/fisiopatología , Animales , Volumen Sanguíneo/efectos de los fármacos , Neoplasias de la Mama/fisiopatología , División Celular/efectos de los fármacos , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Imagen por Resonancia Magnética/métodos , Ratones , Trasplante de Neoplasias , Ratas , Ratas Desnudas , Trasplante Heterólogo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.
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Quinasas de Proteína Quinasa Activadas por Mitógenos , Proteínas Oncogénicas v-mos/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Células 3T3 , Animales , Glutatión Transferasa/metabolismo , MAP Quinasa Quinasa 1 , Ratones , Fosforilación , Proteínas Recombinantes/metabolismo , Serina/metabolismoRESUMEN
The frequency of correctable malocclusion makes it imperative that the condition be diagnosed and referred for appropriate treatment. A complete dentofacial examination, which assesses facial symmetry and the dentition in the transverse, sagittal, and centric relations, is appropriate. Early diagnosis is imperative because malocclusion leads to long-term complications such as temporomandibular joint (TMJ) dysfunctions, perodontal disease, obstructive sleep apnea, psychological disorders, and articulation errors. A combination of orthodontic treatment and orthognathic surgery will correct the dentoskeletal malocclusion and prevent any long-term complications. The purpose of this case report is to describe a patient with severe dentoskeletal malocclusion who benefitted from appropriate orthodontic treatment and orthognathic surgery.
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Maloclusión/diagnóstico , Adulto , Femenino , Humanos , Maloclusión/terapia , Maloclusión de Angle Clase III/complicaciones , Ortodoncia Correctiva , Prognatismo/complicacionesRESUMEN
The displacement of a series of 1,4-benzodiazepine (BDZ) drugs from a chiral stationary phase, based upon human serum albumin, for high performance liquid chromatography was investigated. The different displacement patterns obtained using various mobile phase additives could not be interpreted in terms of binding of the solutes to a single site. The observations were better described by considering the attachment of the BDZs to several loci on the protein. Two main mechanisms of binding were discerned, a nonstereoselective mode, which affected all solutes and seemed to occur at a large number of locations on the protein, and a highly stereoselective mode, which involved only one enantiomer of chiral BDZs and presumably one conformation of certain achiral solutes. The stereoselective binding mode encompassed at least four different sites, each of which displayed slightly different structural requirements. It is suggested that the nomenclature currently used to describe drug binding to human serum albumin may be misleading. Rather than the use of site I or site II, it may be preferable to adopt the terms type I and type II binding, according to the displacement patterns of the compound concerned. This approach would retain the conceptual simplicity of the current notation, while avoiding misleading implications of the exact molecular locus of binding.