RESUMEN
N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.
Asunto(s)
Proteínas de Ciclo Celular/química , Diseño de Fármacos , Pirrolidinonas/síntesis química , Factores de Transcripción/química , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Pirrolidinonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Pulses (grain legumes) are increasingly of interest to the food industry as product formulators and consumers seek to exploit their fiber-rich and protein-rich reputation in the development of nutritionally attractive new products, particularly in the bakery, gluten-free, snack, pasta, and noodle categories. The processing of pulses into consistent high-quality ingredients starts with a well-defined and controlled milling process. However, in contrast to the extensive body of knowledge on wheat flour milling, the peer-reviewed literature on pulse flour milling is not as well defined, except for the dehulling process. This review synthesizes information on milling of leguminous commodities such as chickpea (kabuli and desi), lentil (green and red), pea, and bean (adzuki, black, cowpea, kidney, navy, pinto, and mung) from the perspective of a wheat miller to explore the extent to which pulse milling studies have addressed the objectives of wheat flour milling. These objectives are to reduce particle size (so as to facilitate ingredient miscibility), to separate components (so as to improve value and/or functionality), and to effect mechanochemical transformations (for example, to cause starch damage). Current international standards on pulse quality are examined from the perspective of their relationship to the millability of pulses (that is, grain legume properties at mill receival). The effect of pulse flour on the quality of the products they are incorporated in is examined solely from the perspective of flour quality not quantity. Finally, we identify research gaps where critical questions should be answered if pulse milling science and technology are to be established on par with their wheat flour milling counterparts.
RESUMEN
HIV-1 drug resistance testing in children and adolescents in low-resource settings is both important and challenging. New (more sensitive) drug resistance testing technologies may improve clinical care, but evaluation of their added value is limited. We assessed the potential added value of using next-generation sequencing (NGS) over Sanger sequencing for detecting nucleoside reverse transcriptase inhibitor (NRTI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) drug resistance mutations (DRMs). Participants included 132 treatment-experienced Kenyan children and adolescents with diverse HIV-1 subtypes and with already high levels of drug resistance detected by Sanger sequencing. We examined overall and DRM-specific resistance and its predicted impact on antiretroviral therapy and evaluated the discrepancy between Sanger sequencing and six NGS thresholds (1%, 2%, 5%, 10%, 15%, and 20%). Depending on the NGS threshold, agreement between the two technologies was 62% to 88% for any DRM, 83% to 92% for NRTI DRMs, and 73% to 94% for NNRTI DRMs, with more DRMs detected at low NGS thresholds. NGS identified 96% to 100% of DRMs detected by Sanger sequencing, while Sanger identified 83% to 99% of DRMs detected by NGS. Higher discrepancy between technologies was associated with higher DRM prevalence. Even in this resistance-saturated cohort, 12% of participants had higher, potentially clinically relevant predicted resistance detected only by NGS. These findings, in a young, vulnerable Kenyan population with diverse HIV-1 subtypes and already high resistance levels, suggest potential benefits of more sensitive NGS over existing technology. Good agreement between technologies at high NGS thresholds supports their interchangeable use; however, the significance of DRMs identified at lower thresholds to patient care should be explored further. IMPORTANCE HIV-1 drug resistance in children and adolescents remains a significant problem in countries facing the highest burden of the HIV epidemic. Surveillance of HIV-1 drug resistance in children and adolescents is an important public health strategy, particularly in resource-limited settings, and yet, it is limited due mostly to cost and infrastructure constraints. Whether newer and more sensitive next-generation sequencing (NGS) adds substantial value beyond traditional Sanger sequencing in detecting HIV-1 drug resistance in real life settings remains an open and debatable question. In this paper, we attempt to address this issue by performing a comprehensive comparison of drug resistance identified by Sanger sequencing and six NGS thresholds. We conducted this study in a well-characterized, vulnerable cohort of children and adolescents living with diverse HIV-1 subtypes in Kenya and, importantly, failing antiretroviral therapy (ART) with already extensive drug resistance. Our findings suggest a potential added value of NGS over Sanger even in this unique cohort.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Niño , Humanos , Adolescente , VIH-1/genética , Kenia , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Farmacorresistencia Viral/genética , Genotipo , Carga Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/epidemiología , Mutación , Secuenciación de Nucleótidos de Alto Rendimiento , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéuticoRESUMEN
The sheathing leaf found in grasses and other monocots is an evolutionary innovation, yet its origin has been a subject of long-standing debate. Here, we revisit the problem in the light of developmental genetics and computational modeling. We show that the sheathing leaf likely arose through WOX-gene-dependent extension of a primordial zone straddling concentric domains around the shoot apex. Patterned growth within this zone, oriented by two polarity fields, accounts for wild-type, mutant and mosaic grass leaf development, whereas zone contraction and growth remodeling accounts for eudicot leaf development. In contrast to the prevailing view, our results suggest that the sheath derives from petiole, whereas the blade derives from the lamina of the eudicot leaf, consistent with homologies proposed in the 19th century.
Asunto(s)
Evolución Biológica , Hojas de la Planta/crecimiento & desarrollo , Poaceae/crecimiento & desarrollo , Simulación por Computador , Modelos Biológicos , Morfogénesis , Hojas de la Planta/anatomía & histología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Poaceae/anatomía & histología , Poaceae/genética , Poaceae/metabolismoRESUMEN
N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.
Asunto(s)
Proteínas de Ciclo Celular/antagonistas & inhibidores , Diseño de Fármacos , Pirrolidinonas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Moleculares , Estructura Molecular , Pirrolidinonas/química , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
We investigate, both experimentally and theoretically, the effect of coupling between resonant scatterers on the transmission coefficient of a model system of isotropic scatterers. The model system consists of a monodisperse layer of bubbles, which exhibit a strong monopole scattering resonance at low ultrasonic frequencies. The layer was a true 2D structure obtained by injecting very monodisperse bubbles (with radius a approximately 100 microm) into a yield-stress polymer gel. Even for a layer with a low concentration of bubbles (areal fraction, n pi a(2), of 10-20%, where n is the number of bubbles per unit area), the ultrasonic transmission was found to be significantly reduced by the presence of bubbles (-20 to -50 dB) and showed a sharp minimum at a particular frequency. Interestingly, this frequency did not correspond to the resonance frequency of the individual, isolated bubbles, but depended markedly on the concentration. This frequency shift is an indication of strong coupling between the bubbles. We propose a simple model, based on a self-consistent relation, which takes into account the coupling between the bubbles and gives good agreement with the measured transmission coefficient.
RESUMEN
The primary aim of this review has been to clarify the tumor shrinking effects of dopamine agonists on pituitary macroadenomas of different cell types. Shrinkage is most dramatic for macroprolactinomas and is due to cell size reduction. Seventy-nine percent of 271 definite macroprolactinomas were reduced in size by at least 25%, and 89% shrank to some degree. Most shrinkage occurs during the first 3 months of treatment, although in a minority shrinkage is delayed. Dopamine agonist resistance during long-term therapy is exceptional. Drug withdrawal nearly always leads to a return of hyperprolactinemia, even after several years treatment, although early tumor reexpansion is unusual. About 10% of true macroprolactinomas do not shrink with dopamine agonists; the molecular mechanisms of such resistance have yet to be determined. Alternative formulations of BC and new dopamine agonists (CV 205-502 and cabergoline) are useful for the minority of patients unable to tolerate oral BC, but do not seem to further improve overall shrinkage rates. The risks of pregnancy have probably been overstated, and BC is suitable primary treatment for women with prolactinomas of all sizes; the drug can be used safely during pregnancy in the event of clinically relevant tumor expansion. The interpretation of different degrees of hyperprolactinemia is discussed and management strategies suggested. Most patients with macroprolactinomas now avoid surgery, but drug-induced, time-dependent tumor fibrosis should be remembered if surgery is contemplated. Nonfunctioning pituitary tumors are mostly of gonadotroph cell origin and may be associated with significant disconnection hyperprolactinaemia. Seventy-six of 84 well-characterized tumors showed no tumor shrinkage during dopamine agonist therapy. Possible explanations include abnormalities of dopamine receptor number and function. Preliminary evidence suggests that dopamine agonists may restrain the growth of some functionless tumors; most of these tumors, however, can be satisfactorily debulked using transsphenoidal surgery. In contrast to macroprolactinomas, other functioning pituitary tumors (GH-, TSH-, and ACTH-secreting) rarely shrink during dopamine agonist therapy, although the number of tumors studied is small.
Asunto(s)
Adenoma/tratamiento farmacológico , Dopaminérgicos/uso terapéutico , Neoplasias Hipofisarias/tratamiento farmacológico , Complicaciones Neoplásicas del Embarazo/tratamiento farmacológico , Prolactinoma/tratamiento farmacológico , Adenoma/metabolismo , Adenoma/patología , Bromocriptina/uso terapéutico , División Celular , Femenino , Humanos , Masculino , Metaanálisis como Asunto , Neoplasias Hipofisarias/metabolismo , Embarazo , Complicaciones Neoplásicas del Embarazo/metabolismo , Complicaciones Neoplásicas del Embarazo/patología , Prolactina/sangre , Prolactinoma/metabolismo , Prolactinoma/patologíaRESUMEN
Bubbles, found in a huge variety of food products, are known to afford desirable quality attributes, especially those related to texture, mouthfeel and taste. However, the presence of bubbles and their effects on wheat flour noodles is an aspect that has been, until now, largely overlooked, despite the positive and negative connotations of bubbly inclusions on Asian noodle quality. X-rays from a synchrotron source (Biomedical Imaging and Therapy facility at the Canadian Light Source) were used to rapidly and non-destructively acquire tomographic images of noodle dough. Appropriate image analysis protocols were used to determine the bubble size distribution, the orientation of bubbles, and their position within the dough sheet. The effect of processing (one or multiple lamination steps) on bubble properties in the dough that was subsequently sheeted (gradual elongation and reduction in thickness) was investigated. Bubble size distributions, well captured by lognormal distribution function, showed that the lamination process induced bubble entrapment and reduction in bubble size. Bubbles were found to be flat, elongated and oriented in the sheeting direction, this effect being less for doughs laminated ten times (90° rotations between lamination steps). Interestingly, a gradient in concentration of bubbles within the dough sheet was found from the noodle core to the sheet edges. Aging effects were also apparent. This first non-destructive study of bubbles in wheat-flour noodle dough provides a more complete knowledge of the dough sheet's internal structure, and how it originates via processing, and this has repercussions on the overall quality of Asian noodles.
Asunto(s)
Aire/análisis , Culinaria/métodos , Harina/análisis , Manipulación de Alimentos/métodos , Triticum , Microtomografía por Rayos X , Difusión , SincrotronesRESUMEN
Thyrotropin-releasing hormone immunoreactivity (IR-TRH) has been detected in the circulation of the neonatal rat. This immunoreactivity was demonstrated in purified ethanol extracts of plasma, and was indistinguishable from synthetic TRH using radioimmunoassay and chromatographic criteria. To determine the source of the circulating IR-TRH, tissue concentrations of TRH were analyzed during maturation of the rat. These studies revealed that during the first 10 d of life, the pancreas contained the greatest concentration of IR-TRH of any organ (pancreas, 289+/-35 pg/mg; hypothalamus, 13+/-3 pg/mg, day 5). Thereafter, pancreatic IR-TRH concentrations declined progressively while hypothalamic concentrations gradually increased (pancreas, 1.2+/-0.2 pg/mg; hypothalamus, 365+/-54 pg/mg, adult rat). IR-TRH was also found throughout the gastrointestinal tract but was not detected in the liver, spleen, kidney, or heart. IR-TRH from the pancreas and gastrointestinal tract gave radio-immunoassay binding displacement curves that were parallel to a curve generated with synthetic TRH, and co-migrated with synthetic TRH on Sephadex G-10 and high performance liquid chromatography. In addition, IR-TRH from purified pancreatic extracts was biologically active in that it released thyrotropin and prolactin from rat adenohypophysial cells maintained in monolayer culture. When a total pancreatectomy was performed on the 5th d of life of the rat, mean plasma TRH concentrations were significantly decreased 3 h afterwards (84+/-9 vs. 63+/-7 pg/ml, P < 0.05). Neither the TRH concentrations in the brain, hypothalamus, or gastrointestinal tract, nor the pituitary-thyroid axis were affected by the pancreatectomy. However, mean plasma TRH concentrations remained unaltered 3 h after removal of the hypothalamus and extrahypothalamic brain. FROM THESE RESULTS WE CONCLUDE THE FOLLOWING: (a) the TRH immunoreactivity in the circulation, pancreas, and gastrointestinal tract of the neonatal rat is indistinguishable from synthetic TRH; (b) pancreatic secretion provides a significant contribution to the IR-TRH in plasma, and a proportion of the circulating IR-TRH is derived from other extraneural sites. These findings therefore imply that alterations in hypothalamic and extrahypothalamic brain secretion of TRH are not reflected by changes in levels of this tripeptide in the systemic circulation.
Asunto(s)
Animales Recién Nacidos/sangre , Sistema Digestivo/metabolismo , Páncreas/metabolismo , Hormona Liberadora de Tirotropina/sangre , Factores de Edad , Animales , Encéfalo/metabolismo , Hipotálamo/metabolismo , Pancreatectomía , Radioinmunoensayo , RatasRESUMEN
Ultrasonic spectroscopy is used to characterize a model aerated food system consisting of agar gel in which both bubbles and polystyrene beads are embedded. By exploiting the distinct frequency dependence of each inclusion's acoustic resonances, it is demonstrated that the sizes of the bubbles and beads can be measured by ultrasound even when the size distributions are so similar that these inclusions are difficult to distinguish in optical images. While these results demonstrate the potential for applying ultrasonic spectroscopy to evaluate any soft heterogeneous material in which both bubbles and solid inclusions are present, the technique is especially relevant for functional foods, in which solid functional ingredients must be incorporated without degrading the aerated structure of the food and causing unacceptable quality impairment.
RESUMEN
Pheochromocytoma is rare in pregnancy, with an estimated incidence of 0.007%. Diagnosis is difficult owing to the variety of presentations and nonspecific symptoms. Nevertheless, unsuspected disease accounts for a significant proportion of morbidity and mortality. Currently, there appears to be no consensus on management with regard to the need for and timing of medical vs. surgical management. In this case report, we describe two patients who underwent different modes of treatment based on careful consideration of disease-related and nondisease-related factors. We emphasise that good outcomes can be achieved through individualized management within the context of a multidisciplinary team, involving close collaboration among physicians, surgeons, obstetricians, and anesthetists. We also illustrate the importance of genetic testing in all patients with pheochromocytoma in pregnancy, especially with the emergence of new predisposing genes (succinate dehydrogenase B and D) and the recognition that germline mutations in these and more established genes (VHL and RET) account for over a quarter of all apparently sporadic cases.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/diagnóstico , Neoplasias de las Glándulas Suprarrenales/cirugía , Feocromocitoma/diagnóstico , Feocromocitoma/cirugía , Complicaciones Neoplásicas del Embarazo/diagnóstico , Complicaciones Neoplásicas del Embarazo/cirugía , Resultado del Embarazo , Adulto , Cesárea , Femenino , Humanos , Embarazo , Resultado del TratamientoRESUMEN
Recent papers have explored early events in the development of simple leaves. Functional compartmentalization of the shoot apical meristem correlates with distinct fields of cells connected by plasmodesmata. Molecules important in the initiation of phyllotactic pattern are described and the relationship between dorsoventral patterning and lateral leaf expansion is investigated.
Asunto(s)
Magnoliopsida/crecimiento & desarrollo , Hojas de la Planta/crecimiento & desarrollo , Tipificación del Cuerpo , Linaje de la Célula , Meristema , MorfogénesisRESUMEN
OBJECTIVE: To test the hypothesis whether the effects of GH replacement therapy in adults could be affected by prior pituitary irradiation, the baseline characteristics and response to GH were evaluated in adults with severe GH deficiency (GHD), who had received or not irradiation for the treatment of pituitary adenoma or craniopharyngioma. DESIGN: Data from 447 patients, who had received radiotherapy (427 in addition to surgery), and 630 patients, who were operated on but not irradiated for their tumour, were retrieved from Pfizer International Metabolic Database (KIMS) and compared at baseline and 1 and 2 years following the onset of GH replacement. RESULTS: Irradiated and non-irradiated patients exhibited the expected phenotype of GHD at baseline. However, irradiated patients had a greater impairment in the quality of life (QoL), a higher fat mass, lower high-density lipoprotein cholesterol levels and a lower bone mineral content (BMC) than non-irradiated patients. Treatment with GH induced similar changes in both groups. After 1 year of GH replacement, there was an increase in serum IGF-I and fat-free mass, a reduction in fat mass and an improvement in QoL, all changes being equivalent in irradiated and non-irradiated patients. The lipid profile also improved with the irradiated patients showing a better response. These beneficial effects were maintained and the BMC also increased in both groups by the second year of treatment. CONCLUSIONS: This analysis shows that prior irradiation for pituitary adenoma or craniopharyngioma does not compromise the beneficial effects of GH replacement therapy.
Asunto(s)
Adenoma/radioterapia , Craneofaringioma/radioterapia , Hormona de Crecimiento Humana/administración & dosificación , Hipopituitarismo/tratamiento farmacológico , Neoplasias Hipofisarias/radioterapia , Radioterapia/efectos adversos , Adulto , Bases de Datos Factuales , Femenino , Estudios de Seguimiento , Hormona de Crecimiento Humana/deficiencia , Humanos , Hipopituitarismo/etiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
BACKGROUND: The polypeptide anthopleurin-B (AP-B) is one of a number of related toxins produced by sea anemones. AP-B delays inactivation of the voltage-gated sodium channel of excitable tissue. In the mammalian heart, this effect is manifest as an increase in the force of contraction. As a result, there is interest in exploiting the anthopleurins as lead compounds in the design of novel cardiac stimulants. Essential to this endeavour is a high-resolution solution structure of the molecule describing the positions of functionally important side chains. RESULTS: AP-B exists in multiple conformations in solution as a result of cis-trans isomerization about the Gly40-Pro41 peptide bond. The solution structure of the major conformer of AP-B has been determined by two-dimensional 1H NMR at pH 4.5 and 25 degrees C. The core structure is a four-stranded, antiparallel beta-sheet (residues 2-4, 20-23, 34-37 and 45-48) and includes several beta-turns (6-9, 25-28, 30-33). Three loops connect the beta-strands, the longest and least well defined being the first loop, extending from residues 8-17. These features are shared by other members of this family of sea anemone toxins. The locations of a number of side chains which are important for the cardiac stimulatory activity of AP-B are well defined in the structures. CONCLUSIONS: We have described the solution structure of AP-B and compared it with that of AP-A, from which it differs by substitutions at seven amino acid positions. It shares an essentially identical fold with AP-A yet is about 10-fold more active. Comparison of the structures, particularly in the region of residues essential for activity, gives a clearer indication of the location and extent of the cardioactive pharmacophore in these polypeptides.
Asunto(s)
Cardiotónicos/química , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Gráficos por Computador , Glicina , Enlace de Hidrógeno , Péptidos y Proteínas de Señalización Intercelular , Isomerismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Prolina , Anémonas de Mar , Soluciones , Relación Estructura-ActividadRESUMEN
BACKGROUND: The ornamental tobacco Nicotiana alata produces a series of proteinase inhibitors (PIs) that are derived from a 43 kDa precursor protein, NaProPI. NaProPI contains six highly homologous repeats that fold to generate six separate structural domains, each corresponding to one of the native PIs. An unusual feature of NaProPI is that the structural domains lie across adjacent repeats and that the sixth PI domain is generated from fragments of the first and sixth repeats. Although the homology of the repeats suggests that they may have arisen from gene duplication, the observed folding does not appear to support this. This study of the solution structure of a single NaProPI repeat (aPI1) forms a basis for unravelling the mechanism by which this protein may have evolved. RESULTS: The three-dimensional structure of aPI1 closely resembles the triple-stranded antiparallel beta sheet observed in each of the native PIs. The five-residue sequence Glu-Glu-Lys-Lys-Asn, which forms the linker between the six structural domains in NaProPI, exists as a disordered loop in aPI1. The presence of this loop in aPI1 results in a loss of the characteristically flat and disc-like topography of the native inhibitors. CONCLUSIONS: A single repeat from NaProPI is capable of folding into a compact globular domain that displays native-like PI activity. Consequently, it is possible that a similar single-domain inhibitor represents the ancestral protein from which NaProPI evolved.
Asunto(s)
Nicotiana/genética , Proteínas de Plantas/química , Plantas Tóxicas , Inhibidores de Proteasas/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/genéticaRESUMEN
BACKGROUND: kappa-PVIIA is a 27-residue polypeptide isolated from the venom of Conus purpurascens and is the first member of a new class of conotoxins that block potassium channels. By comparison to other ion channels of eukaryotic cell membranes, voltage-sensitive potassium channels are relatively simple and methodology has been developed for mapping their interactions with small-peptide toxins. PVIIA, therefore, is a valuable new probe of potassium channel structure. This study of the solution structure and mode of channel binding of PVIIA forms the basis for mapping the interacting residues at the conotoxin-ion channel interface. RESULTS: The three-dimensional structure of PVIIA resembles the triple-stranded beta sheet/cystine-knot motif formed by a number of toxic and inhibitory peptides. Subtle structural differences, predominantly in loops 2 and 4, are observed between PVIIA and other conotoxins with similar structural frameworks, however. Electrophysiological binding data suggest that PVIIA blocks channel currents by binding in a voltage-sensitive manner to the external vestibule and occluding the pore. Comparison of the electrostatic surface of PVIIA with that of the well-characterised potassium channel blocker charybdotoxin suggests a likely binding orientation for PVIIA. CONCLUSIONS: Although the structure of PVIIA is considerably different to that of the alphaK scorpion toxins, it has a similar mechanism of channel blockade. On the basis of a comparison of the structures of PVIIA and charybdotoxin, we suggest that Lys19 of PVIIA is the residue which is responsible for physically occluding the pore of the potassium channel.
Asunto(s)
Conotoxinas , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Canales de Potasio/química , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Electrofisiología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Venenos de Moluscos/síntesis química , Oocitos/fisiología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Unión Proteica , Estructura Secundaria de Proteína , Soluciones , Xenopus laevisRESUMEN
By transfecting the adenovirus 5 E1A gene into neu-transformed NIH3T3 cells, we previously showed that E1A can dramatically repress neu-encoded p185 expression and, concomitantly, suppress the features of transformation and metastasis of neu+E1A transfectants. From these results we concluded that suppression of transformation and metastasis by E1A in neu-transformed cells may be through repression of neu gene expression. However, E1A has recently been shown to also repress the transformation features of other human cancer cells that do not overexpress neu. This observation raised a possibility that repression of neu gene expression in our neu+E1A cells might not be the only mechanism for transformation and metastasis suppression. To study whether other molecular mechanisms might be involved in suppression of transformation and metastasis by E1A in our neu+E1A cells, we reexpressed p185 oncoprotein in the neu+E1A cells by transfecting them with a plasmid containing activated rat neu complementary DNA and we examined whether E1A can suppress transformation and metastasis when the neu-encoded p185 protein is reexpressed. All the features of transformed cells including cell morphology, DNA synthesis rate, colony formation in soft agar, and tumorigenicity in nu/nu mice were restored in the cell lines that reexpressed neu. In addition, the levels of neu reexpression corresponded to the degree of malignant transformation. However, the in vivo metastatic tumor formation by these p185 reexpressing cells was still significantly inhibited by E1A. When metastasis-associated properties were examined in the cell lines that reexpressed p185, we found that cell motility was recovered by reexpression of p185 to the degree corresponding to the p185 reexpression level, but secretion of membrane-degrading gelatinases and invasion through the basement membrane preparation Matrigel by these cells were still inhibited by E1A. The data demonstrated that reexpression of p185 in neu+E1A cells can counteract the tumor-suppressing function of E1A but not completely recover the neu-induced metastatic phenotype. We conclude from these results that (a) repression of neu oncogene expression was indeed the molecular mechanism by which E1A suppressed tumor formation in neu-transformed 3T3 cells, and (b) suppression of metastasis by E1A in neu-transformed 3T3 cells was via multiple molecular mechanisms in addition to repressing neu. Our model system clearly demonstrated that tumorigenicity and metastasis are related but separable phenomena.
Asunto(s)
Proteínas E1A de Adenovirus/fisiología , Transformación Celular Neoplásica/genética , Receptores ErbB/análisis , Metástasis de la Neoplasia/genética , Proteínas Proto-Oncogénicas/análisis , Células 3T3 , Proteínas E1A de Adenovirus/análisis , Animales , Línea Celular , Receptores ErbB/genética , Gelatinasas/metabolismo , Ratones , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2RESUMEN
Amplification or overexpression of c-erbB-2/neu protooncogene, or both, occur frequently in many different types of human cancers and have been shown to correlate with decreased survival in ovarian cancer patients. We have previously found that the ovarian carcinoma cell line SK-OV-3 overexpresses c-erbB-2/neu mRNA. To further study the biological effect of c-erbB-2/neu overexpression in SK-OV-3 cells, we injected such cells i.p. into female nu/nu mice and found that this cell line forms extensive abdominal tumors and ascites. From the ascites in an injected mouse, we established the SKOV3.ip1 cell line and found that it expressed 2-fold more c-erbB-2/neu-encoded p185 proteins than the parental SK-OV-3 cells. When transformation phenotypes of SK-OV-3 and SKOV3.ip1 cells were compared, SKOV3.ip1 cells showed higher cell growth and DNA synthesis rates, formed more colonies in soft agar, produced larger s.c. tumors, and resulted in shorter survival of nu/nu mice after i.p. injection. These data indicate that the level of c-erbB-2/neu overexpression may correlate with the degree of malignancy in these ovarian carcinoma cells. Since we had previously shown that the adenovirus 5 E1A gene product can suppress transformation and metastatic properties induced by mutation-activated rat neu oncogene in mouse embryo fibroblast cells, we further examined whether E1A can abrogate malignancy in c-erbB-2/neu-overexpressing human ovarian cancer cells. We introduced the E1A gene into c-erbB-2/neu-overexpressing SKOV3.ip1 cells and found that the E1A-expressing ovarian cancer cell lines had decreased c-erbB-2/neu-encoded p185 expression and reduced malignancy, including a decreased ability to induce tumors in nu/nu mice. Therefore, we concluded that E1A is a tumor suppressor gene for c-erbB-2/neu-overexpressing human ovarian cancer cells and may be useful in developing therapeutic reagents for these human cancers.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor/genética , Genes Virales , Neoplasias Ováricas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transfección , Proteínas E1A de Adenovirus/biosíntesis , Animales , División Celular , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2 , Células Tumorales Cultivadas , Ensayo de Tumor de Célula MadreRESUMEN
In the present study, the influence of octenyle succinic anhydride (OSA),gum concentration, pH, temperature and reaction time on esterification of Persian gum (PG), and its soluble (SFPG) and insoluble (IFPG) fractions, were investigated by response surface methodology (RSM) in order to optimize the reaction conditions based on the degree of substitution (DS). The individual effect of all independent variables as well as the interactive effects of temperature-OSA concentration, and OSA-PG concentrations on DS was significant. However, the latter interactive effect (OSA-SFPG) was not significant in case of SFPG. The IFPG did not have any esterification reaction with OSA. The highest DS for PG and SFPG were 0.0285 and 0.0303 at the optimal conditions, respectively. The FTIR spectrums also confirmed the carbonyl group attachment in OSA-PG and OSA-SFPG. The enhancement of emulsifying capability was also confirmed by ECI and EAI values, microscopic images as well as rheological measurements.
Asunto(s)
Gomas de Plantas/química , Succinatos/química , Anhídridos Succínicos/química , Emulsionantes/química , Esterificación , Reología , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Almidón/químicaRESUMEN
kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in kappa-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.