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This study explores how adolescents engage with unhealthy food and beverage marketing in online settings, from a gender perspective. Employing an online ethnography approach and using go-along interviews, we explored the experiences of adolescent boys and girls aged 13-17 as they navigated their online experiences with digital food and beverage marketing. Notable themes emerged, including the identification of predatory actions by food companies, the role of protective factors such as family, and the influence of social media influencers in shaping adolescent dietary preferences. Importantly, this research unearthed gender disparities in the participants' responses. Girls, in particular, exhibited a heightened awareness of the protective role played by their families, emphasized the influence of color in marketing strategies, recognized the significance of gender in marketing, and reported exposure to alcohol advertisements-findings that boys less frequently echoed. The study underscores the importance of adolescence as a critical phase in development, during which food companies target these impressionable individuals, driven by their independence and potential for brand loyalty. Moreover, it highlights the potential avenue of gender-specific marketing, offering valuable insights into the gendered dimensions of adolescents' food marketing experiences. By examining the interplay between digital food marketing and gender, this research addresses a critical gap in the literature, shedding light on how gender influences adolescents' perceptions, responses, and behaviors in the context of food marketing strategies. These findings have the potential to inform adolescents of the marketing techniques that target them and guide policymakers in developing and implementing evidence-based regulations aimed at safeguarding adolescents from exposure to unhealthy food marketing.
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Alimentos , Mercadotecnía , Masculino , Femenino , Adolescente , Humanos , Factores Sexuales , Mercadotecnía/métodos , Bebidas , Dieta , Industria de AlimentosRESUMEN
INTRODUCTION: The floating catchment area (FCA) method has emerged as the most comprehensive and accurate method for quantifying the spatial accessibility of health care services. There were variants of the FCA-based method that was continuously improvised by the researchers to suit specific local contexts and the different nature of healthcare service delivery. This scoping review identifies factors associated with the spatial accessibility of healthcare services that were specifically measured using the FCAbased method. MATERIALS AND METHODS: This scoping review was performed through electronic databases (PubMed and ScienceDirect) using keywords: 'spatial accessibility', 'floating catchment area' and 'factors'. Google Scholar and Mendeley Network were also used as additional sources to obtain relevant studies. RESULTS: A total of 32 articles were included in this review. Factors identified can be distinguished into two broad categories, which are spatial and non-spatial factors. Spatial factors were remoteness or distance from the urban centre, areas in close proximity to main roads, and some specific geographical characteristics such as mountainous and deltaic regions, whereas non-spatial factors were the degree of urbanisation, population density and various demographic profiles of the population such as socioeconomic status, health need, and minority ethnic composition. CONCLUSION: This study adds to the body of literature pertinent to the factors associated with spatial accessibility to healthcare services. These findings could give insight for researchers to consider and incorporate those additional variables to further improve the FCA-based method calculations.
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Áreas de Influencia de Salud , Accesibilidad a los Servicios de Salud , HumanosRESUMEN
Background With optimum transfusion and chelation therapy, the survival of ß-thalassemia patients and the incidence of various complications, including renal complications, have improved. Objectives To investigate renal involvement in ß-thalassemia patients using serum and urinary biochemical markers of glomerular and tubular dysfunction. Methods This case-control study included 69 ß-thalassemia major (ß-TM) patients, 23 ß-thalassemia intermedia (ß-TI) patients, and 100 healthy controls, all ranging from 1 to 16 years in age. Blood urea nitrogen (BUN), serum ferritin, serum and urinary levels of creatinine (Cr), uric acid (UA), calcium (Ca), phosphorus (Ph), magnesium (Mg), sodium (Na), and potassium (K), and the urinary albumin/creatinine ratio were evaluated. Results The BUN level and the urinary Na/Cr, K/Cr, Ca/Cr, Mg/Cr, Ph/Cr, albumin/Cr, and UA/Cr ratios were significantly higher in the ß-thalassemia patients than in the controls. In contrast, the serum Na, K, Ca, and Mg levels were significantly lower in the patients (P<0.05). An elevated urinary UA/Cr ratio was found in 61.9% of ß-thalassemia patients, and an elevated urinary Ca/Cr, and urinary albumin/Cr ratio was found in 53.2%. An elevated Na/Cr ratio was found in 41.3%. The serum and urinary renal markers showed no significant differences between patients with ß-TM and ß-TI, except for microscopic hematuria, which was significantly higher in ß-TI patients (34.8%) than in ß-TM patients (13%), P>0.02. At an older age, high serum ferritin levels and deferoxamine therapy were associated with significant tubular and glomerular dysfunction in ß-thalassemia patients. Conclusions Pediatric patients with ß-thalassemia have significantly abnormal tubular and glomerular functions, necessitating early detection and monitoring to prevent/reverse renal function deterioration.
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The interaction of mineral nutrients with metals/metalloids and signalling molecules is well known. In the present study, we investigated the effect of phosphorus (P) in mitigation of arsenic (As) stress in mustard (Brassica juncea L.). The study was conducted to investigate potential of 30 mg P·kg-1 soil P supplement (diammonium phosphate) to cope up with the adverse effects of As stress (24 mg As·kg-1 soil) in mustard plants Supplementation of P influenced nitric oxide (NO) generation, which up-regulated proline metabolism, ascorbate-glutathione system and glyoxalase system and alleviated the effects of on photosynthesis and growth. Arsenic stress generated ROS and methylglyoxal content was scavenged through P-mediated NO, and reduced As translocation from roots to leaves. The involvement of NO under P-mediated alleviation of As stress was substantiated with the use of cPTIO (NO biosynthesis inhibitor) and SNP (NO inducer). The reversal of P effects on photosynthesis under As stress with the use of cPTIO emphasized the role of P-mediated NO in mitigation of As stress and protection of photosynthesis The results suggested that P reversed As-induced oxidative stress by modulation of NO formation, which regulated antioxidant machinery. Thus, P-induced regulatory interaction between NO and reversal of As-induced oxidative stress for the protection of photosynthesis may be suggested for sustainable crops.
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Arsénico , Planta de la Mostaza , Antioxidantes , Arsénico/toxicidad , Suplementos Dietéticos , Óxido Nítrico , Estrés Oxidativo , Fósforo , FotosíntesisRESUMEN
OBJECTIVES: An optimal gestational weight gain is essential for maternal health and to reduce adverse birth outcomes. Current guidelines to monitor gestational weight gain are based on pre-pregnancy body mass index (BMI). However, middle-upper arm circumference (MUAC) is increasingly used as an alternative nutritional status measure for pregnant women. Hence, this study aimed to determine associations of MUAC and pre-pregnancy BMI with gestational weight gain rate among Malaysian pregnant women. STUDY DESIGN: A cross-sectional study was conducted among 444 pregnant women (≥20â¯weeks gestation). MAIN OUTCOMES MEASURES: Women completed questionnaires on sociodemographic data, maternal characteristics and pre-pregnancy weight. Height, current weight and MUAC were measured at study visit (from 1st February 2016 to 31st January 2017). RESULTS: About a third (34.24%) of pregnant women were overweight or obese prior to pregnancy. MUAC was inversely associated with an inadequate rate of gestational weight gain (ORâ¯=â¯0.77; 95% CI: 0.68, 0.87) as compared to normal gestational weight gain. In contrast, a higher MUAC was associated with a higher odds ratio (ORâ¯=â¯1.28; 95% CI: 1.11, 1.49) of having excessive rate of gestational weight. No associations were found for pre-pregnancy BMI categories for gestational weight gain rate. CONCLUSION: Our findings revealed that women with low MUAC were more likely to have an inadequate gestational weight gain rate during pregnancy whereas higher MUAC was associated with an excessive gestational weight gain rate. MUAC may be a useful indicator of nutritional status associated with GWG. Routine measurement of MUAC in pregnant women may help health professionals, particularly in middle-income countries, to counsel women about gestational weight gain.
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Brazo/anatomía & histología , Índice de Masa Corporal , Ganancia de Peso Gestacional , Adulto , Estudios Transversales , Femenino , Humanos , Malasia , Estado Nutricional , Obesidad/fisiopatología , Embarazo , Encuestas y Cuestionarios , Adulto JovenRESUMEN
BACKGROUND: A global rise in multidrug-resistant (MDR) nosocomial infections has led to a significant increase in morbidity and mortality. MDR Gram-negative bacteria (GNB) are recognised for rapidly developing drug resistance. Despite Pseudomonas aeruginosa being the second most common GNB isolated from healthcare associated infections, the magnitude of MDR P. aeruginosa (MDR-PA) has not been evaluated in Qatar. AIM: To assess the prevalence and antimicrobial susceptibility patterns of MDR-PA from 5 major hospitals in Qatar. METHODS: A total of 2533 P. aeruginosa clinical isolates were collected over a one-year period. MDR-PA was defined as resistance to at least one agent of ≥ 3 antibiotic classes. Clinical and demographic data were collected prospectively. FINDINGS: The overall prevalence of MDR-PA isolates was 8.1% (205/2533); the majority of isolates were from patients exposed to antibiotics during 90 days prior to isolation (85.4 %, 177/205), and the infections were mainly hospital-acquired (95.1%, 195/205) with only 4.9% from the community. The majority of MDR-PA isolates were resistant to cefepime (96.6%, 198/205), ciprofloxacin, piperacillin/tazobactam (91%, 186/205), and meropenem (90%, 184/205). Patient comorbidities with MDR-PA were diabetes mellitus (47.3%, n=97), malignancy (17.1%, n=35), end-stage renal disease (13.7%, n=28) and heart failure (10.7%, n=22). CONCLUSION: There was a significant prevalence of MDR-PA in Qatar, primarily from healthcare facilities and associated with prior antibiotic treatment. There was an alarming level of antimicrobial resistance to carbapenems. Our results are part of a national surveillance of MDR to establish effective containment plans.
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Osteoporosis is affecting over 200 million people worldwide. Despite available guidelines, care for these patients remains sub-optimal. We developed an osteoporosis tool to address the multiple dimensions of chronic disease management. Findings from its evaluation showed a significant increase from baseline in osteoporosis investigations and treatment, so we are revising this tool to include multiple chronic conditions including an update of evidence about osteoporosis. Our objectives were to conduct a systematic review of osteoporosis interventions in adults at risk for osteoporosis. We searched bibliometric databases for randomized controlled trials (RCTs) in any language evaluating osteoporosis disease management interventions in adults at risk for osteoporosis. Reviewer pairs independently screened citations and full-text articles, extracted data, and assessed risk of bias. Analysis included random effects meta-analysis. Primary outcomes were osteoporosis investigations and treatment, and fragility fractures. Fifty-five RCTs and one companion report were included in the analysis representing 165,703 patients. Our findings from 55 RCTs and 18 sub-group meta-analyses showed that complex implementation interventions with multiple components consisting of at least education + feedback + follow-up significantly increased the initiation of osteoporosis medications, and interventions with at least education + follow-up significantly increased the initiation of osteoporosis investigations. No significant impact was found for any type of intervention to reduce fracture. Complex interventions that include at least education + follow-up or feedback have the most potential for increasing osteoporosis investigations and treatment. Patient education appears to be an important component in osteoporosis disease management.
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Osteoporosis/diagnóstico , Osteoporosis/terapia , Densidad Ósea , Conservadores de la Densidad Ósea/uso terapéutico , Manejo de la Enfermedad , Utilización de Medicamentos/estadística & datos numéricos , Humanos , Osteoporosis/fisiopatología , Fracturas Osteoporóticas/fisiopatología , Fracturas Osteoporóticas/prevención & control , Educación del Paciente como Asunto/estadística & datos numéricosRESUMEN
Objective Malnutrition in children pervades all aspects of their health, growth, cognitive and social development and can lead to irreversible and lifelong effects. We examine the prevalence and determinants of malnutrition in children under 5 in the Ghanaian population. Methods Using data from the latest available Ghana Demographic and Health Survey (GDHS), we estimated and compared prevalence of malnutrition in children among the different subgroups of the population. We used multivariable logistic regression to identify potential factors associated with childhood malnutrition in Ghana. Results Overall, 35.6 % (95 % CI: 33.6, 37.6) of Ghanaian children under 5 years of age suffer from some form of malnutrition. Specifically, 27.5 % (95 % CI: 25.1, 28.7), 13.8 % (95 % CI: 12.5, 15.3), 8.9 % (95 % CI: 7.8, 10.2) were stunted, underweight and wasted, respectively. Results from the logistic regression indicate that gender and age of the child, educational and nutritional status of the mother, and financial status of the household are risk factors associated with childhood malnutrition in Ghana. Conclusions for Practice In view of the observed high rate of malnutrition among Ghanaian children despite the interventions that have been in place since the 1990s, there is a need for increased awareness and improved targeted interventions as well as knowledge translation tools including extensive education on infant and young child feeding practices.
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Lactancia Materna/métodos , Cuidado del Lactante/métodos , Desnutrición/epidemiología , Desnutrición/prevención & control , Madres/educación , Adulto , Factores de Edad , Preescolar , Femenino , Geografía , Ghana/epidemiología , Humanos , Lactante , Recién Nacido , Modelos Logísticos , Masculino , Prevalencia , Factores Sexuales , Factores SocioeconómicosRESUMEN
PURPOSE: Evidence regarding the relationship between red blood cell methotrexate polyglutamate concentration and response to treatment and adverse drug reactions in patients using methotrexate for inflammatory arthropathies is complex and in some respects appears conflicting. Accordingly, we undertook a systematic analysis of available evidence to determine the clinical utility of dosing methotrexate to a target red blood cell methotrexate polyglutamate concentration. METHODS: A systematic literature review was conducted to identify all studies that had reported an association between red blood cell methotrexate polyglutamate concentration and disease activity or adverse drug reactions in users of methotrexate for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis or psoriatic arthritis. RESULTS: No randomised controlled trials were identified. Thirteen studies (ten in patients with rheumatoid arthritis and three in patients with juvenile idiopathic arthritis) were identified. All studies evaluated an association between red blood cell methotrexate polyglutamate concentration and response to treatment, and eight evaluated an association with toxicity. Eight studies identified lower disease activity with at least one higher red blood cell methotrexate polyglutamate concentration, although there was at least moderate potential for bias in all of these studies. Relatively large increases in concentration appeared to be required to produce a meaningful reduction in disease activity. Only one study identified an association between red blood cell methotrexate polyglutamate concentration and methotrexate-induced side effects, although studies were likely underpowered to detect this type of association. CONCLUSIONS: The manner in which data were presented in the included studies had many limitations that hampered its conclusive assessment, but red blood cell methotrexate polyglutamate concentrations appear to be a potentially useful guide to treatment in patients with inflammatory arthropathies, but the specific polyglutamate that should be monitored and how monitoring could be integrated into treat-to-target approaches should be clarified before it can be routinely implemented.
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Antirreumáticos/metabolismo , Antirreumáticos/uso terapéutico , Artritis/tratamiento farmacológico , Artritis/metabolismo , Eritrocitos/metabolismo , Metotrexato/análogos & derivados , Ácido Poliglutámico/análogos & derivados , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antirreumáticos/efectos adversos , Niño , Ensayos Clínicos como Asunto , Estudios Transversales , Relación Dosis-Respuesta a Droga , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/metabolismo , Femenino , Humanos , Masculino , Metotrexato/efectos adversos , Metotrexato/metabolismo , Metotrexato/uso terapéutico , Persona de Mediana Edad , Ácido Poliglutámico/efectos adversos , Ácido Poliglutámico/metabolismo , Ácido Poliglutámico/uso terapéutico , Estudios ProspectivosRESUMEN
BACKGROUND: Few studies have studied the role of homocysteine in migraineurs and have produced conflicting results. The MTHFR C677T genotype has been associated with increased risk of migraine in selected clinical samples. We assessed the association of the MTHFR C677T variant with migraine, the corresponding homocysteine levels and their correlation. METHOD: We studied 27 random adult migraineurs with aura (MWA), migraine without aura (MWOA), and 32 non-migraineurs (controls) from Lahore, Pakistan in this pilot study which is still under progress. RESULTS: We found significant differences in homocysteine levels between various diagnostic groups (K-W test: p=0.005). One-way ANOVA, post-hoc tests revealed significant differences in homocysteine levels between Non-migraineurs, MWA (p=0.002, CI: 1.93 - 9.19) and MWoA (p=0.002, CI: -9.19 - -1.9). We found a significant association between the migraine group and C677T-MTHFR variant mutant allele (C/T) (p=0.039). We did not find a significant association between C677T-MTHFR variant and homocysteine levels. CONCLUSION: In this pilot study, we found plasma homocysteine levels to be significantly associated with MWOA. Additionally, plasma homocysteine levels were lower in MWA than in MWOA. Furthermore, we did not find a relationship between homocysteine levels and the MTHFR variant (SNP rs1801133). Lastly, there may be a relationship between the MTHFR variant (SNP rs1801133) and migraine in this population.
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Homocisteína/sangre , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Migraña sin Aura/sangre , Migraña sin Aura/genética , Adulto , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , MasculinoRESUMEN
We have investigated the channel structural determinants that underlie the difference in gating properties of Cav3.1 and Cav3.3 T-type channels, by creating a series of chimeric channel constructs in which the major transmembrane domains were swapped. The chimeras were then expressed in tsA-201 cells and subjected to whole cell patch clamp analysis. Our data reveal that domains I and IV are major determinants of the half-activation potential. Substitution of domain IV was the most important determinant of activation time constant and time constant for recovery from inactivation, with domains I and II mediating a smaller role. In contrast, the carboxy terminal region did not appear to be involved. Determinants of the time constant for inactivation could not be localized to a specific transmembrane domain, but the concomitant substitution of domains I+IV was able to partially confer the inactivation kinetics among the two wild type channels. Our data indicate that the domain IV region mediates an important role in T-type channel activation, whereas multiple channel structural determinants appear to control T-type channel inactivation.
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Canales de Calcio Tipo T/fisiología , Activación del Canal Iónico/fisiología , Proteínas de Transporte de Membrana/fisiología , Canales de Calcio Tipo T/genética , Línea Celular Transformada , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica/métodos , Humanos , Activación del Canal Iónico/efectos de la radiación , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Proteínas de Transporte de Membrana/genética , Mutagénesis/fisiología , Proteínas Mutantes Quiméricas/fisiología , Técnicas de Placa-Clamp/métodos , Estructura Terciaria de Proteína/fisiología , Transfección/métodosRESUMEN
Coexistence of sickle cell disease (SCD) and systemic lupus erythematosus (SLE) has been reported in 11 patients. The authors describe five additional patients with SCD and symptoms initially attributable to SCD who were later found to have SLE. Patients were identified over a 10-year period (1991-2001) in a pediatric sickle cell population numbering approximately 350. All patients are African-American. Age at diagnosis of SLE was 9 to 17 years (median 11 years), and follow-up after diagnosis ranged from 6 months to 10 years (median 3 years). SLE cerebritis (n = 3), serositis (n = 4), and nephritis (n = 2) were common findings. Physicians should be alerted to the possible development of SLE in patients with SCD.
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Anemia de Células Falciformes/complicaciones , Población Negra , Lupus Eritematoso Sistémico/etiología , Adolescente , Adulto , Edad de Inicio , Niño , Femenino , Humanos , Lupus Eritematoso Sistémico/patología , Masculino , Estudios RetrospectivosRESUMEN
Calcium channels are important regulators of neuronal excitability and contribute to transmitter release, calcium dependent gene expression, and oscillatory behavior in many cell types. Under physiological conditions, native low-voltage (T-type)- and high-voltage-activated (HVA) currents are potently inhibited by trivalent cations. However, the presence of multiple calcium channel isoforms has hampered our ability to unequivocally assess the effects of trivalent cations on channel activity. Here, we describe the actions of nine trivalent metal ions on transiently expressed alpha1G (Cav3.1) T-type calcium channels cloned from human brain. In 2 mM external barium solution, yttrium most potently inhibited alpha1G current (IC50 = 28 nM), followed by erbium > gadolinium ~ cerium > holmium > ytterbium > neodymium > lanthanum >> scandium. With the exception of scandium, blocking affinity was loosely correlated with decreasing ionic radius. A detailed characterization of yttrium block revealed a 25-fold decrease in blocking affinity when the external concentration of charge carrier was increased from 2 mM to 20 mM. In 20 mM barium, yttrium also effectively inhibited various types of cloned HVA channels indicating that this ion is a nonselective blocker. For all calcium channels examined, yttrium preferentially inhibited inward over outward current, but block was otherwise voltage independent. In addition to peak current inhibition, P/Q- and L-type channels underwent a unique speeding of the macroscopic time course of inactivation. Whereas peak current block of alpha1A channels was highly sensitive to the external charge carrier concentration, the inactivation effects mediated by yttrium were not, suggesting that the two effects are due to distinct mechanisms. Moreover, the speeding effect was greatly attenuated by manipulations that slowed the inactivation kinetics of the channels. Thus, our evidence suggests that yttrium effects are mediated by two distinct events: peak current block likely occurring by occlusion of the pore, and kinetic speeding arising from yttrium interactions with the channel that alter the state of the inactivation gate.
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Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Cationes/farmacología , Metales/farmacología , Canales de Calcio/clasificación , Células Cultivadas , Cerio/farmacología , Erbio/farmacología , Gadolinio/farmacología , Holmio/farmacología , Humanos , Riñón/embriología , Riñón/fisiología , Lantano/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neodimio/farmacología , Escandio/farmacología , Iterbio/farmacología , Itrio/farmacologíaRESUMEN
We recently reported that amino acid residues contained within a putative EF hand motif in the domain III S5-H5 region of the alpha(1B) subunit affected the relative barium:calcium permeability of N-type calcium channels (Feng, Z. P., Hamid, J., Doering, C., Jarvis, S. E., Bosey, G. M., Bourinet, E., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 5726-5730). Since this region partially overlaps with residues previously implicated in block of the channel by omega-conotoxin GVIA, we assessed the effects of mutations in the putative EF hand domain on channel block by omega-conotoxin GVIA and the structurally related omega-conotoxin MVIIA. Both of the toxins irreversibly block the activity of wild type alpha(1B) N-type channels. We find that in addition to previously identified amino acid residues, residues in positions 1326 and 1332 are important determinants of omega-conotoxin GVIA blockade. Substitution of residue Glu(1332) to arginine slows the time course of development of block. Point mutations in position Gly(1326) to either arginine, glutamic acid, or proline dramatically decrease the time constant for development of the block. Additionally, in the G1326P mutant channel activity was almost completely recovered following washout. A qualitatively similar result was obtained with omega-conotoxin MVIIA, suggesting that common molecular determinants underlie block by these two toxins. Taken together the data suggest that residue Gly(1326) may form a barrier, which controls the access of peptide toxins to their blocking site within the outer vestibule of the channel pore and also stabilizes the toxin-channel interaction.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/fisiología , Glicina , omega-Conotoxina GVIA/farmacología , omega-Conotoxinas/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina , Sitios de Unión , Calcio/metabolismo , Canales de Calcio Tipo N/efectos de los fármacos , Línea Celular , Ácido Glutámico , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Prolina , Estructura Secundaria de Proteína , Subunidades de Proteína , TransfecciónRESUMEN
It is widely believed that the selectivity of voltage-dependent calcium channels is mainly controlled by amino acid residues contained within four p-loop motifs forming the pore of the channel. An examination of the amino acid sequences of high voltage-activated calcium channels reveals that their domain III S5-H5 regions contain a highly conserved motif with homology to known EF hand calcium binding proteins, hinting that this region may contribute to channel permeation. To test this hypothesis, we used site-directed mutagenesis to replace three conserved negatively charged residues in the N-type calcium channel alpha1B subunit (Glu-1321, Asp-1323, and Glu-1332) with positively charged amino acids (lysine and arginine) and studied their effect on ion selectivity using whole cell and single channel patch clamp recordings. Whereas the wild type channels conducted barium much more effectively than calcium, the mutant displayed nearly equal permeabilities for these two ions. Individual replacement of residue 1332 or a double substitution of residues 1321 and 1323 with lysine and arginine, respectively, were equally effective. Disruption of the putative EF hand motif through replacement of the central glycine residue (1326) with proline resulted in a similar effect, indicating that the responses observed with the triple mutant were not due to changes in the net charge of the channel. Overall, our data indicate that residues outside of the narrow region of the pore have the propensity to contribute to calcium channel permeation. They also raise the possibility that interactions of calcium ions with a putative calcium binding domain at the extracellular side of the channel may underlie the differential permeabilities of the channel for barium and calcium ions.
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Canales de Calcio Tipo N/metabolismo , Calcio/metabolismo , Motivos EF Hand , Transporte Biológico , Canales de Calcio Tipo N/genética , Conductividad Eléctrica , Glicina/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Mutación Puntual , Isoformas de ProteínasRESUMEN
The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the alpha(1B) calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein beta subunits on both wild type and mutant alpha(1B) calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of Gbeta(1) on channel activity, whereas Gbeta(2), Gbeta(3), and Gbeta(4) are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the Gbeta(1) subunit.
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Canales de Calcio Tipo N/metabolismo , Proteínas de Unión al GTP/fisiología , Proteína Quinasa C/fisiología , Proteínas de Unión al GTP/química , Humanos , Fosforilación , Isoformas de Proteínas/fisiología , Subunidades de Proteína , Transmisión SinápticaRESUMEN
We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/fisiología , Animales , Encéfalo/metabolismo , Canales de Calcio/efectos de los fármacos , Línea Celular , Ácido Egtácico/farmacología , Humanos , Riñón , Sustancias Macromoleculares , Potenciales de la Membrana/fisiología , Modelos Moleculares , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Tetraetilamonio/farmacologíaRESUMEN
Voltage operated calcium channels (VOCCs) are implicated in osteoblastic mechano- and hormonal transduction. Very little, however, is known about the expression of VOCCs in osteoblasts of load-bearing bones. Here we describe two types of whole-cell calcium current in rat femoral explant-derived osteoblasts. The first is high-voltage activated and sensitive to nifedipine, Bay K8644 and FPL 64176. The second is low-voltage activated and is sensitive to micromolar concentrations of Ni2+. The properties of these two currents are consistent with those of L-type and T-type calcium currents respectively. T-type currents were detected in most cells on the day of passage, the level of expression being significantly lower on subsequent days. L-type currents were also most common on the day of passage but were detected consistently throughout the 4-day period of study. The reverse transcription polymerase chain reaction with non-specific primers directed against all L-type VOCC alpha 1 subunits and then with specific primers directed against sequences from rat brain alpha 1C (L-type), alpha 1D (L-type) and alpha 1G (T-type) VOCC subunits detected transcripts of appropriate size in all four cases. Products from the three sets of specific primer pairs (alpha 1C, alpha 1D, alpha 1G) were sequenced and were identical to their respective rat brain templates.
Asunto(s)
Huesos/fisiología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo T/metabolismo , Osteoblastos/metabolismo , Soporte de Peso/fisiología , Animales , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/fisiología , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/fisiología , Células Cultivadas , Conductividad Eléctrica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The modulation of presynaptic calcium channel activity by second messengers provides a fine tuning mechanism for neurotransmitter release. In neurons, the activation of certain G protein-coupled receptors reduces N-type channel activity by approximately 60%. In contrast, activation of protein kinase C (PKC) results in an approximately 50% increase in N-type channel activity, and subsequent G protein inhibition is antagonized. Here, we describe the molecular determinants that control the dual effects of PKC-dependent phosphorylation. The double substitution of two adjacent PKC consensus sites in the calcium channel domain I-II linker (Thr422, Ser425) to alanines abolished both PKC-dependent up-regulation and the PKC-G protein cross-talk. The single substitution of Ser425 to glutamic acid abolished PKC up-regulation but had no effect on G protein modulation. Replacement of Thr422 with glutamic acid eliminated PKC-dependent up-regulation and mimicked the effects of PKC phosphorylation on G protein inhibition. Our data suggest that Thr422 mediates the antagonistic effect of PKC on G protein modulation, while phosphorylation of either Thr422 or Ser425 are sufficient to increase N-type channel activity. Thus, Thr422 serves as a molecular switch by which PKC is able to simultaneously trigger the up-regulation of channel activity and antagonize G protein inhibition.