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The consumption of poultry products contaminated with Salmonella species is one of the most common causes of Salmonella infections. In vivo studies demonstrated the potential application of peanut skins (PS) as an antimicrobial poultry feed additive to help mitigate the proliferation of Salmonella in poultry environments. Tons of PS, a waste by-product of the peanut industry, are generated and disposed in U.S. landfills annually. Peanut skins and extracts have been shown to possess antimicrobial and antioxidant properties. Hence, we aimed to determine the effect of PS as a feed additive on the gut microbiota of broilers fed a control or PS supplemented (4% inclusion) diet and inoculated with or without Salmonella enterica Enteritidis (SE). At hatch 160 male broilers were randomly assigned to 4 treatments: 1) CON-control diet without SE, 2) PS-PS diet without SE, 3) CONSE-control diet with SE, 4) PSSE-PS diet with SE. On d 3, birds from CONSE and PSSE treatments were inoculated with 4.2 × 109 CFU/mL SE. At termination (4 wk), 10 birds/treatment were euthanized and ileal and cecal contents were collected for 16S rRNA analysis using standard methodologies. Sequencing data were analyzed using QIIME2. No effect of PS or SE was observed on ileal alpha and beta diversity, while evenness, richness, number of amplicon sequence variants (ASV) and Shannon, as well as beta diversity were significantly (P < 0.05) affected in ceca. Similarly, more differentially abundant taxa between treatment groups were identified in ceca than in ileum. However, more microbiota functional changes, based on the PICRUST2 prediction, were observed in ileum. Overall, relatively minor changes in microbiota were observed during SE infection and PS treatment, suggesting that PS addition may not attenuate the SE proliferation, as shown previously, through modulation of microbiota in gastrointestinal tract. However, while further studies are warranted, these results suggest that PS may potentially serve as a functional feed additive for poultry for improvement of animal health.
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The design of next-generation alloys through the integrated computational materials engineering (ICME) approach relies on multiscale computer simulations to provide thermodynamic properties when experiments are difficult to conduct. Atomistic methods such as density functional theory (DFT) and molecular dynamics (MD) have been successful in predicting properties of never before studied compounds or phases. However, uncertainty quantification (UQ) of DFT and MD results is rarely reported due to computational and UQ methodology challenges. Over the past decade, studies that mitigate this gap have emerged. These advances are reviewed in the context of thermodynamic modeling and information exchange with mesoscale methods such as the phase-field method (PFM) and calculation of phase diagrams (CALPHAD). The importance of UQ is illustrated using properties of metals, with aluminum as an example, and highlighting deterministic, frequentist, and Bayesian methodologies. Challenges facing routine uncertainty quantification and an outlook on addressing them are also presented.
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We aimed to determine the effects of feeding a high-oleic peanut (HOPN) diet to egg-producing laying hens on egg quality, digestibility, and feed conversion. Three isonitrogenous and isocaloric dietary treatments were formulated with 1) Control diet (CON)-a corn-soybean meal conventional diet with 7.8 % added poultry fat, 2) HOPN diet-dietary inclusion of ~20% coarse-ground whole HOPN, and 3) oleic acid (CON-OA) diet-a control diet supplemented with 2.6% oleic fatty acid oil. Ninety-nine 57-wk-old brown Leghorn laying hens were randomly assigned to 33 animals per treatment. Animals were housed individually for 8 wk. Body and feed weights were recorded weekly and feed conversation ratio was calculated. Bi-weekly, shell eggs were analyzed for quality (yolk color, albumen height, and Haugh unit [HU]). Jejunum samples were collected at week 8 for histomorphometric analysis. Analysis of variance was performed on all variables using a general linear mixed model. Laying hens fed the CON-OA diet produced greater number of eggs relative to those fed the HOPN and control diets (P < 0.05). The roche yolk color value was higher (P < 0.001) in eggs from hens fed the HOPN diet. There were no differences in laying hen performance, eggshell color, eggshell strength, eggshell elasticity and egg albumen height, or egg HU, ileal fat digestibility, or villi surface among treatment groups. However, the apparent metabolizable energy (P < 0.01) and ileal protein digestibility (P = 0.02) were greater in laying hens fed the HOPN diet relative to the CON diet. This study suggests that whole unblanched high-oleic peanuts may be an acceptable alternative feed ingredient for laying hens.
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BACKGROUND AND AIM: Chronic subordinate colony housing (CSC, 19 days), an established and preclinically-validated mouse model for posttraumatic stress disorder (PTSD), causes evening hypocorticism and a reduced adrenal in vitro ACTH (adrenocorticotropic hormone) sensitivity despite pronounced adrenal hyperplasia. However, until now it remains unclear at what time point during CSC exposure evening hypocorticism and adrenal in vitro ACTH insensitivity develop and whether the repeated change of dominant aggressor mice plays an important role in this context. It is, therefore, the aim of the current study, to explore the detailed time course of these stress-induced adrenal changes. METHODS: Adrenal weight, plasma corticosterone (CORT) and ACTH were assessed in the morning of days 8 (right before exposure to the 2nd aggressor), 9 (24 h after exposure to the 2nd aggressor), 15 (right before exposure to the 3rd aggressor), 16 (24 h after exposure to the 3rd aggressor) and 20 or in the evening of days 8 (10 h after exposure to the 2nd aggressor), 9 (34 h after exposure to the 2nd aggressor), 15 (10 h after exposure to the 3rd aggressor), 16 (34 h after exposure to the 3rd aggressor) and 20 of CSC exposure. Moreover, we in vitro cultured adrenal explants of all mice euthanized in the morning of days 8, 9, 15, 16 and 20 either in the presence or absence of ACTH to subsequently assess CORT concentration in the supernatants. RESULTS: Our results indicate that while adrenal mass was increased at all time points assessed, plasma morning CORT only transiently increased in response to the 2nd (on day 8) but not 3rd (on day 15) dominant aggressor mouse. Moreover, although mild signs of adrenal in vitro ACTH insensitivity developed already after one week of CSC exposure, moderate and severe adrenal in vitro ACTH insensitivity required two and three weeks of chronic subordination, respectively. CONCLUSION: Together with unaffected plasma ACTH levels at all time points assessed, our data suggest that stress-induced adrenal in vitro ACTH insensitivity develops gradually during times of chronic subordination while subordination to different aggressor mice aggravates its severity. Moreover, a mild form of adrenal ACTH insensitivity seems to allow prevention of morning hypercorticism on day 8 of CSC, despite functional adrenal mass being increased, while a moderate and severe form of adrenal ACTH insensitivity in CSC mice seems to promote HPA axis adaptation to repeated homotypic stressor exposure (i.e. dominant aggressor mice) and basal evening hypocorticism in CSC mice, respectively. Our results might, therefore, be the basis for future clinical studies assessing CORT supplementation as novel treatment regimen for somatic and affective pathologies linked to chronic and/or traumatic stress.
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Glándulas Suprarrenales/metabolismo , Glándulas Suprarrenales/fisiología , Estrés Psicológico/metabolismo , Hormona Adrenocorticotrópica/sangre , Animales , Conducta Animal/fisiología , Corticosterona/sangre , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Sistema Hipófiso-Suprarrenal/metabolismo , Conducta SocialRESUMEN
Locally grown feed ingredients of high energy and protein content, such as peanuts, maybe economically feasible alternatives to corn and soybean meal in broiler diets. Even though normal-oleic peanuts have been demonstrated to be a viable feed ingredient for poultry, few studies to date have examined the use of high-oleic peanuts (HO PN) as an alternative feed ingredient for broiler chickens. Thus, we aimed to determine the effect of feeding HO PN on broiler performance, nutrient digestibility, and intestinal morphology. Three isocaloric, isonitrogenous experimental diets were formulated with 1) dietary inclusion of ~10% coarse-ground whole HO PN; 2) a corn-soybean meal control diet with 5.5% added poultry fat; and 3) a control diet supplemented with 5.5% oleic fatty acid oil. Three-hundred Ross 708 broilers were randomly placed in 10 replicate pens per treatment with 10 chicks per pen and raised until 42 d. Body weights (BW) and feed intake were determined weekly, and feed conversion ratio (FCR) was calculated. Jejunum samples were collected at 42 d for histomorphometric analysis. Analysis of variance was performed on all variables using a general linear mixed model in JMP Pro14. Broilers in the HO PN group had lower (P < 0.05) BW and higher FCR than other treatment groups at weeks 2 and 6. There were no significant differences in the jejunum villi surface area between the treatment groups. However, broilers fed the HO PN diet had greater (P = 0.019) apparent metabolizable energy relative to the other treatment groups, suggesting improved nutrient uptake of dietary fats and/or carbohydrates in the HO PN treatment group. However, additional studies are warranted to further define the nutritional value of HO PN as an alternative poultry feed ingredient.
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Increasing biopotency of cholecalciferol (D3) from vitamin sources is essential in the poultry industry to meet nutritional demands and counter stressors. D3 exhibits hormonal traits and is responsible for calcium (Ca) absorption. 1-α-Hydroxycholecalciferol (1α) is a synthetic form of D3 that has equal efficacy and is cheaper to synthesize than 1,25-dihydroxycholecalciferol (active form of D3), on broilers. However, 1α bypasses a critical regulatory point, the kidney, and may consequently lead to toxicity levels of Ca via Ca absorption. This study examined 1α supplementation in broiler diets with different Ca inclusion levels to determine if 1α at higher Ca levels caused Ca toxicity at starter and grower phases with Ross 708 male broiler chicks. In Experiment 1 (1-15 days of age), chicks were assigned to one of 10 treatment starter diets with five levels of Ca inclusion (0.80, 0.95, 1.10, 1.25, and 1.40%) with or without 1α supplementation (5 µg 1α/kg in feed) and eight replicate cages per treatment. In Experiment 2, chicks were fed common starter diet until 16 days of age, and then they were assigned to one of eight treatment diets with four levels of Ca inclusion (0.54, 0.76, 0.98, or 1.20%) with or without 1α supplementation (5 µg 1α/kg in feed). At the end of both experiments, blood was collected from broilers to determine blood chemistry, including concentrations of vitamin D metabolites. Intestinal tissues were also collected to examine gene expression. In Experiment 1, broilers not fed 1α exhibited a quadratic effect in ionized blood Ca (iCa) as dietary Ca inclusion levels increased; 1α-fed broilers displayed an increase in iCa as Ca inclusion levels increased (p = 0.0002). For Experiment 2, 1α-fed broilers displayed a decrease in 25-hydroxycholecalciferol plasma concentration as dietary Ca inclusion levels increased (p = 0.035); also, increasing Ca inclusion in diets increased expression of duodenal sodium phosphate cotransporter type II b (NPTIIb, p = 0.03). Our findings imply that inclusion of 1α was beneficial because 1α enhanced Ca absorption during the starter phase; however, to avoid potential Ca toxicity or antagonism while using 1α during the grower phase, there should be consideration with reducing dietary Ca levels.
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Previous studies have demonstrated that allergenic feed proteins from peanuts in the diets of layer hens are not detected in the eggs produced. Hence, in this study, we aimed to determine if soy and/or peanut proteins in poultry feed rations of broiler chickens or layer hens would be transferred or detectable in the meat or eggs produced. To meet this objective, 99 layer hens and 300 broiler chickens were equally divided into treatment groups and fed one of three experimental diets: control soybean meal and corn diet, whole unblanched high-oleic peanut and corn diet (HO PN), or a control diet spiked supplemented with oleic acid (OA) oil. At termination, broiler chickens were processed, and chicken breast samples of the left pectoralis muscle were collected, and eggs were collected from layers. Total protein extracts from pooled egg samples and chicken breast samples were subjected to enzyme-linked immunosorbent assay (ELISA) methods and immunoblotting analysis with rabbit antipeanut agglutinin antibodies and rabbit antisoy antibodies for the detection of peanut and soy proteins. Peanut and soy proteins were undetected in all pooled egg samples and individual chicken breast meat samples using immunoblotting techniques with rabbit antipeanut agglutinin and rabbit antisoy antibodies. Moreover, quantitative ELISA allergen detection methods determined all pooled egg samples and individual meat samples as "not containing" peanut or soy allergens. Therefore, this study helps to evaluate the risk associated with the potential transfer of allergenic proteins from animal feed to the products produced for human consumption.
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Previous studies have identified peanut meal prepared from normal-oleic peanuts as a suitable and economical ingredient for broiler feed. However, to date, no studies have examined the use of new, high-oleic peanut (HO-PN) cultivars as a feed ingredient for poultry. This project aimed to determine the effect of HO-PNs as a feed ingredient for broiler chickens on the quality and sensory attributes of the meat produced. To test 3 experimental diets, male broiler chicks were randomly placed, at hatch, in raised-wire cages, in 10 replicate pens per treatment with 10 chicks per cage. For 6 wk, chicks were fed, ad libitum, one of the three isocaloric, isonitrogenous diets: (1) a conventional soybean meal plus corn control diet, (2) 10 to 12% HO-PN + corn diet, or (3) a control corn diet spiked with ≈6.0% oleic fatty acid oil (OA). At 42 D, 3 broilers per pen (30 per treatment) were processed to determine meat quality and for consumer evaluation. Carcass weights and breast yields were reduced in broilers fed HO-PN, while leg carcass yields were greater in broilers fed HO-PN in comparison to the other groups. Chicken breast from broilers fed HO-PN had reduced meat-pH, reduced L* color values, and increased cooked loss compared to other treatments. Nevertheless, a group of 100-consumer panelists scored all 3-treatment groups similar in terms of sensory attributes for cooked chicken. While additional studies must be performed, this study suggest that HO-PN may be a suitable broiler feed ingredient.
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Alimentación Animal/análisis , Arachis/química , Carne/análisis , Nueces/química , Ácido Oléico/análisis , Animales , Pollos , Dieta/veterinaria , Masculino , Distribución AleatoriaRESUMEN
Auxin is a major phytohormone that controls numerous aspects of plant development and coordinates plant responses to the environment. Morphogenic gradients of auxin govern cell fate decisions and underlie plant phenotypic plasticity. Polar auxin transport plays a central role in auxin maxima generation. The discovery of the exquisite spatiotemporal expression patterns of auxin biosynthesis genes of the WEI8/TAR and YUC families suggested that local auxin production may contribute to the formation of auxin maxima. Herein, we systematically addressed the role of local auxin biosynthesis in plant development and responses to the stress phytohormone ethylene by manipulating spatiotemporal patterns of WEI8. Our study revealed that local auxin biosynthesis and transport act synergistically and are individually dispensable for root meristem maintenance. In contrast, flower fertility and root responses to ethylene require local auxin production that cannot be fully compensated for by transport in the generation of morphogenic auxin maxima.
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Ácidos Indolacéticos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/embriología , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Etilenos/metabolismo , Flores/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Meristema/embriología , Meristema/metabolismo , Morfogénesis , Oxigenasas , Desarrollo de la Planta/genética , Reguladores del Crecimiento de las Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Análisis Espacio-TemporalRESUMEN
The quest towards expansion of the M n+1AX n design space has been accelerated with the recent discovery of several solid solution and ordered phases involving at least two M n+1AX n end members. Going beyond the nominal M n+1AX n compounds enables not only fine tuning of existing properties but also entirely new functionality. This search, however, has been mostly done through painstaking experiments as knowledge of the phase stability of the relevant systems is rather scarce. In this work, we report the first attempt to evaluate the finite-temperature pseudo-binary phase diagram of the Ti2AlC-Cr2AlC via first-principles-guided Bayesian CALPHAD framework that accounts for uncertainties not only in ab initio calculations and thermodynamic models but also in synthesis conditions in reported experiments. The phase stability analyses are shown to have good agreement with previous experiments. The work points towards a promising way of investigating phase stability in other MAX Phase systems providing the knowledge necessary to elucidate possible synthesis routes for M n+1AX n systems with unprecedented properties.
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This article provides a broad overview regarding intent to initiate and consider ongoing chronic opioid therapy (COT) for treatment of chronic noncancer pain (CNCP). COT should be an individualized decision based on a comprehensive evaluation, assessment, and monitoring. It is imperative that providers discuss various risks and benefits of COT initially and at follow-up visits, and continue appropriate monitoring and follow-up at regular intervals. The decision to initiate or continue opioid therapy is based on clinical judgment; however, it is understood that opioid and other medication therapy represent one piece of the complete treatment plan for patients with CNCP.
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Analgésicos Opioides/uso terapéutico , Analgésicos/uso terapéutico , Dolor Crónico/tratamiento farmacológico , Atención Primaria de Salud/organización & administración , Dolor Crónico/psicología , Relación Dosis-Respuesta a Droga , Medicina Basada en la Evidencia , Humanos , Dimensión del Dolor/métodos , Relaciones Médico-PacienteRESUMEN
BACKGROUND: Methadone has been a stalwart pharmacologic option for the management of opioid drug dependence for many years. It substitutes for opioid agonists and possesses certain pharmacokinetic properties that confer characteristics preferable to those of other opioids for this application. Methadone is likewise used as an option for the treatment of pain, particularly chronic pain. It has a spectrum of pharmacodynamic activity, including contributions from non-opioid components, that translates to its specific clinical attributes as an analgesic. Unfortunately, basic science studies and accumulated clinical experience with methadone have revealed some undesirable, and even worrisome, features, including issues of safety. The benefit/risk ratio of methadone might be acceptable if there was no better alternative, but neither its pharmacokinetic nor pharmacodynamic properties are unique to methadone. OBJECTIVE: We review the basic and clinical pharmacology of methadone and suggest that levorphanol should receive attention as a possible alternative. CONCLUSION: Unlike methadone, levorphanol is a more potent NMDA antagonist, possesses a higher affinity for DOR and KOR, has a shorter plasma half-life yet longer duration of action, has no CYP450 interactions or QTc prolongation risk, can be a viable option in the elderly, palliative care, and SCI patients, requires little to no need for co-administration of adjuvant analgesics, and has potentially a lower risk of drug-related Emergency Department visits compared to other opioids.
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Analgésicos Opioides/uso terapéutico , Levorfanol/uso terapéutico , Metadona/uso terapéutico , Manejo del Dolor/métodos , Analgésicos Opioides/farmacocinética , Humanos , Levorfanol/farmacocinética , Metadona/farmacocinéticaRESUMEN
BACKGROUND: There is a need to have a model to study methadone's losses during hemodialysis to provide informed methadone dose recommendations for the practitioner. AIM: To build a one-dimensional (1-D), hollow-fiber geometry, ordinary differential equation (ODE) and partial differential equation (PDE) countercurrent hemodialyzer model (ODE/PDE model). METHODOLOGY: We conducted a cross-sectional study in silico that evaluated eleven hemodialysis patients. Patients received a ceiling dose of methadone hydrochloride 30 mg/day. Outcome measures included: the total amount of methadone removed during dialysis; methadone's overall intradialytic mass transfer rate coefficient, km ; and, methadone's removal rate, j ME. Each metric was measured at dialysate flow rates of 250 mL/min and 800 mL/min. RESULTS: The ODE/PDE model revealed a significant increase in the change of methadone's mass transfer with increased dialysate flow rate, %Δkm =18.56, P=0.02, N=11. The total amount of methadone mass transferred across the dialyzer membrane with high dialysate flow rate significantly increased (0.042±0.016 versus 0.052±0.019 mg/kg, P=0.02, N=11). This was accompanied by a small significant increase in methadone's mass transfer rate (0.113±0.002 versus 0.014±0.002 mg/kg/h, P=0.02, N=11). The ODE/PDE model accurately predicted methadone's removal during dialysis. The absolute value of the prediction errors for methadone's extraction and throughput were less than 2%. CONCLUSION: ODE/PDE modeling of methadone's hemodialysis is a new approach to study methadone's removal, in particular, and opioid removal, in general, in patients with end-stage renal disease on hemodialysis. ODE/PDE modeling accurately quantified the fundamental phenomena of methadone's mass transfer during hemodialysis. This methodology may lead to development of optimally designed intradialytic opioid treatment protocols, and allow dynamic monitoring of outflow plasma opioid concentrations for model predictive control during dialysis in humans.
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As with other South Asian countries, injury is becoming a leading cause of death and morbidity among children in Vietnam. In response to the increasing burden of child injury, government and non-government agencies in Vietnam have combined efforts during the last decade to develop and implement various child injury prevention strategies and programmes. This article provides, through a review of relevant documents and interviews with major stakeholders, an overview of these efforts and highlights major challenges to child injury prevention in the country. The findings point to notable achievements in terms of increasing awareness of injury facing children at all levels in the community and developing a sound injury prevention policy framework in a relatively short period of time. However, much needs to be done to implement necessary environmental and legislative changes, strengthen child injury surveillance and injury prevention research; and to improve access to health services. The insight into the experience of Vietnam could benefit other low- and middle-income countries with a high burden of child injury.
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Países en Desarrollo , Educación en Salud , Política Pública , Heridas y Lesiones/prevención & control , Niño , Ahogamiento/prevención & control , Planificación Ambiental , Dispositivos de Protección de la Cabeza , Humanos , Motocicletas/legislación & jurisprudencia , Natación/educación , VietnamRESUMEN
OBJECTIVES: The study aims to determine the incidence of suicide attempt, describe the methods used, and assess use of health care services including mental health care after suicide attempt in a rural area of Vietnam. METHODS: All suicide attempters (104) during 2003-2007 were listed, diagnosed and re-evaluated by trained physicians according to the research criteria of the WHO Multicentre Study of Attempted Suicide. All attempters were interviewed by trained medical staff to investigate methods used, socio-demographic characteristics and use of health services. RESULTS: The yearly incidence was 10.2 per 100000 person-years, 10.6 per 100000 in males and 9.8 per 100000 in females. 99% of cases committed suicide attempt by poisoning, 62.6% by pesticides and 36.3% by pharmaceutical drugs. 34.3% reported having been in contact with somatic care and 13.2% had received mental health care. Among those who reported some treatment received, 47.5% had been in contact with official health care services, 8.1% had pharmacy keepers' consultation or were treated by traditional healers and 4% reported self treatment. CONCLUSION: The incidence of suicide attempt was lower in this population compared to other settings. While the majority of attempters use pesticides, many had used psychotropic drugs. Contact with mental health services following the attempt was very limited in this setting. Suicide prevention for this high risk group should focus on reducing access to pesticides and psychotropic drugs. Mental health services should be made more accessible in rural areas.
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BACKGROUND: We present a case of spinal leptomeningeal metastasis from an intracranial glioblastoma multiforme that presented with radicular pain. CASE DESCRIPTION: A 55-year-old man with a previously treated supratentorial glioblastoma multiforme presented with a 12-month history of thoracic radicular pain. MRI of the thoracic spine demonstrated an intradural extramedullary metastatic tumor deposit at the levels of T8-T10. External beam radiotherapy to the thoracic spine provided a minimal decrease in the intensity of the radicular pain. The lack of appreciation of the metastatic potential of the primary intracranial tumor resulted in delayed diagnosis and treatment. CONCLUSION: Spinal leptomeningeal metastasis needs to be suspected in patients with a past history of intracranial glioblastoma multiforme, who present with the clinical features of radiculopathy or myelopathy. Awareness of this condition will facilitate appropriate intervention.
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Glioblastoma/secundario , Neoplasias Meníngeas/secundario , Radiculopatía/etiología , Neoplasias de la Médula Espinal/secundario , Neoplasias Supratentoriales/diagnóstico , Diagnóstico Diferencial , Glioblastoma/diagnóstico , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Imagen por Resonancia Magnética , Masculino , Neoplasias Meníngeas/diagnóstico , Neoplasias Meníngeas/patología , Neoplasias Meníngeas/radioterapia , Persona de Mediana Edad , Radiculopatía/patología , Radiculopatía/radioterapia , Médula Espinal/patología , Neoplasias de la Médula Espinal/diagnóstico , Neoplasias de la Médula Espinal/patología , Neoplasias de la Médula Espinal/radioterapia , Neoplasias Supratentoriales/patología , Neoplasias Supratentoriales/radioterapia , Lóbulo Temporal/patologíaRESUMEN
Responses to extracellular stimuli are often transduced from cell-surface receptors to protein tyrosine kinases which, when activated, initiate the formation of protein complexes that transmit signals throughout the cell. A prominent component of these complexes is the product of the proto-oncogene c-Cbl, which specifically targets activated protein tyrosine kinases and regulates their signalling. How, then, does this multidomain protein shape the responses generated by these signalling complexes?
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Proteínas de Caenorhabditis elegans , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Actinas/metabolismo , Animales , Citoesqueleto/metabolismo , Regulación hacia Abajo , Inducción Embrionaria , Proteínas del Helminto/metabolismo , Humanos , Ratones , Ratones Noqueados , Modelos Moleculares , Proteína Oncogénica v-cbl , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Oncogénicas de Retroviridae/química , Proteínas Oncogénicas de Retroviridae/deficiencia , Proteínas Oncogénicas de Retroviridae/genética , Proteínas Oncogénicas de Retroviridae/metabolismo , Transducción de Señal , Linfocitos T/fisiología , Ubiquitinas/metabolismoRESUMEN
The c-Cbl protooncogene can function as a negative regulator of receptor protein tyrosine kinases (RPTKs) by targeting activated receptors for polyubiquitination and downregulation. This function requires its tyrosine kinase binding (TKB) domain for targeting RPTKs and RING finger domain to recruit E2 ubiquitin-conjugating enzymes. It has therefore been proposed that oncogenic Cbl proteins act in a dominant-negative manner to block this c-Cbl activity. In testing this hypothesis, we found that although mutations spanning the RING finger abolish c-Cbl-directed polyubiquitination and downregulation of RPTKs, they do not induce transformation. In contrast, it is mutations within a highly conserved alpha-helical structure linking the SH2 and RING finger domains that render Cbl proteins oncogenic. Thus, Cbl transformation involves effects additional to polyubiquitination of RPTKs that are independent of the RING finger and its ability to recruit E2-conjugating enzymes.
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Transformación Celular Neoplásica , Regulación hacia Abajo , Receptores ErbB/metabolismo , Mutación/genética , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas , Ubiquitinas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Endocitosis , Humanos , Ligasas/metabolismo , Ratones , Datos de Secuencia Molecular , Proteína Oncogénica pp60(v-src)/metabolismo , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-cbl , Alineación de Secuencia , Enzimas Ubiquitina-Conjugadoras , Dedos de Zinc/genética , Dominios Homologos src/genéticaRESUMEN
Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation. We show that Cbl overexpression in 293T embryonic kidney and Jurkat T-lymphocyte cells led to a dramatic reduction in the active pool of Fyn; this was seen as a reduction in Fyn autophosphorylation, reduced phosphorylation of in vivo substrates, and inhibition of transcription from a Src-family kinase response element linked to a luciferase reporter. Importantly, a Fyn mutant (FynY528F) relieved of intramolecular repression was still negatively regulated by Cbl. The Cbl-dependent negative regulation of Fyn did not appear to be mediated by inhibition of Fyn kinase activity but was correlated with enhanced protein turnover. Consistent with such a mechanism, elevated levels of Fyn protein were observed in cell lines derived from Cbl(-/-) mice compared to those in wild-type controls. The effects of Cbl on Fyn were not observed when the 70ZCbl mutant protein was analyzed. Taken together, these observations implicate Cbl as a component in the negative regulation of Fyn and potentially other Src-family kinases, especially following kinase activation. These results also suggest that protein degradation may be a general mechanism for Cbl-mediated negative regulation of activated tyrosine kinases.