How Europeans move: a moderate-to-vigorous physical activity and sitting time paradox in the European Union.

OBJECTIVES: This study aimed to assess the interactions between physical activity (PA) and sedentary behaviour in a large population taking account of major sociodemographic characteristics. STUDY DESIGN: Cross-sectional population-based study. METHODS: Data from 28,031 individuals living in the European Union who were aged ≥15 years were retrieved from a cross-sectional survey, the Eurobarometer 2017. Interactions among the four mobility components (vigorous, moderate, walking activity and sitting time) were assessed at the individual level across age, gender and place of residence, and at the country level by compositional data analysis, hierarchical linear regressions and principal component analysis. RESULTS: The most frequently reported PA was walking; however, sitting time represented >95% of the reported weekly times, whereas moderate-to-vigorous PA (MVPA) represented <1%. Women reported less PA and sitting time, age decreased total PA and increased sitting time, and individuals living in large urban areas reported lower PA and higher sitting times. MVPA decreased with age (ß = -0.047, P < 0.001) and was lower in women (ß = -0.760, P < 0.001) and those living in large urban areas (ß = -0.581, P < 0.001), while walking and sitting times increased with age, being higher in women and lower in those living in rural areas. At the country level, sitting time was positively associated with moderate activity (ß = 0.389, P = 0.041) and marginally non-significant with MVPA (ß = 0.330, P = 0.087). CONCLUSIONS: Walking was the highest contributor to weekly PA, whereas sitting time was paradoxically associated with higher MVPA. Specific measures to reduce sitting time are required to achieve an active lifestyle.

Colorenal fistula and urinary tract infection in a pediatric patient with acute lymphoblastic leukemia. / Fístula colorrenal e infección de vías urinarias en paciente pediátrico con leucemia linfoide aguda.

INTRODUCTION: Colorenal fistula is rare in the pediatric population. It may occur at any segment involved by ischemia, chronic inflammation, or necrosis. It is typically associated with a preliminary renal lesion that may arise as a result of interventional procedures, inflammatory conditions, colon tumor, and xanthogranulomatous pyelonephritis, among others. CASE REPORT: 15-year-old female patient diagnosed with acute lymphoblastic leukemia admitted at our institution for baseline condition management. During her stay, she experienced gastrointestinal and urinary infectious events. In the assessment and management of those, a left colorenal fistula was found. Surgical treatment was decided upon. DISCUSSION: Colorenal fistula typically occurs secondary to renal inflammation or infection. Clinical signs are highly variable, and treatment is surgical, with the fistulous tract being resected in all cases.

INTRODUCCION: Las fístulas colorrenales son infrecuentes en la población pediátrica. Pueden desarrollarse en cualquier segmento afectado por isquemia, inflamación crónica o necrosis. Suelen estar asociadas a una lesión primitiva en el riñón que puede producirse por procedimientos intervencionistas, enfermedades inflamatorias, tumorales del colon, pielonefritis xantogranulomatosa, entre otras. CASO CLINICO: Paciente femenina de 15 años, con diagnóstico de leucemia linfoide aguda, ingresa a la institución para recibir manejo de su enfermedad de base. Durante su evolución, desarrolla eventos infecciosos (gastrointestinales y urinarios), y en evaluación y manejo de estos se documenta fístula colorrenal izquierda, motivo por el cual se da un enfoque de tratamiento quirúrgico. COMENTARIOS: La fístula renocólica generalmente se presenta secundaria a procesos inflamatorios o infecciosos renales; su presentación clínica es muy variada, y el tratamiento es quirúrgico, incluyendo siempre la resección del trayecto fistuloso.

Fístula colorrenal e infección de vías urinarias en paciente pediátrico con leucemia linfoide aguda / Colorenal fistula and urinary tract infection in a pediatric patient with acute lymphoblastic leukemia

Introducción: Las fístulas colorrenales son infrecuentes en la población pediátrica. Pueden desarrollarse en cualquier segmento afectadopor isquemia, inflamación crónica o necrosis. Suelen estar asociadas auna lesión primitiva en el riñón que puede producirse por procedimientos intervencionistas, enfermedades inflamatorias, tumorales del colon,pielonefritis xantogranulomatosa, entre otras. Caso clínico: Paciente femenina de 15 años, con diagnóstico deleucemia linfoide aguda, ingresa a la institución para recibir manejode su enfermedad de base. Durante su evolución, desarrolla eventosinfecciosos (gastrointestinales y urinarios), y en evaluación y manejode estos se documenta fístula colorrenal izquierda, motivo por el cualse da un enfoque de tratamiento quirúrgico. Comentarios: La fístula renocólica generalmente se presenta secundaria a procesos inflamatorios o infecciosos renales; su presentaciónclínica es muy variada, y el tratamiento es quirúrgico, incluyendo siempre la resección del trayecto fistuloso.(AU)

Introduction: Colorenal fistula is rare in the pediatric population.It may occur at any segment involved by ischemia, chronic inflammation, or necrosis. It is typically associated with a preliminary renallesion that may arise as a result of interventional procedures, inflammatory conditions, colon tumor, and xanthogranulomatous pyelonephritis,among others. Clinical case: 15-year-old female patient diagnosed with acutelymphoblastic leukemia admitted at our institution for baseline condition management. During her stay, she experienced gastrointestinal andurinary infectious events. In the assessment and management of those,a left colorenal fistula was found. Surgical treatment was decided upon. Discussion: Colorenal fistula typically occurs secondary to renalinflammation or infection. Clinical signs are highly variable, and treatment is surgical, with the fistulous tract being resected in all cases.(AU)

Management of the neovascular choroidal membrane secondary to ocular toxoplasmosis. / Manejo de la membrana neovascular coroidea secundaria a toxoplasmosis ocular.

A 12-year-old patient diagnosed with congenital toxoplasmosis, with no systemic treatment at the time, who presented with a decreased visual acuity (VA) in his left eye (LE). On examination, VA in the LE was 0.05 and the fundus examination revealed a focus of chorioretinitis adjacent to a pigmented macular scar, as well as a large associated subretinal haemorrhage. After confirming the diagnosis of choroidal neovascular membrane secondary to ocular toxoplasmosis, treatment was started with systemic anti-toxoplasmosis drugs and two anti-VEGF intravitreal injections separated by one month. Finally, the patients had a VA in LE of 0.4, with reabsorption of the haemorrhage, leaving an inactive pigmented macular scar. The use of anti-VEGF intravitreal injections in cases of ocular toxoplasmosis has been associated with a reactivation of old lesions, so the prophylactic use of oral anti-toxoplasmosis drugs is recommended in these cases.

Acute narrow-angle glaucoma induced by topiramate with acute myopia and macular striae: A case report. / Glaucoma agudo de ángulo cerrado inducido por topiramato con miopización aguda y estrías maculares: a propósito de un caso.

We report the case of a 29-year-old epileptic woman who had been on treatment with topiramate 25mg/day for 9 days. She was referred to the Emergency Department due to reduction in far visual acuity (VA) after increasing the dose to 50mg/day two days before. The ocular examination showed bilateral acute angle closure glaucoma (AACG) and macular striae in both eyes (AO) observed by Retinography and Optical Coherence Tomography (OCT). The AACG is a well-known side effect of topiramate, but the macular striae rarely accompanies it. Although macular striae have been previously described in other cases, very few document those using retinography and OCT images. Therefore, it is important to differentiate a case of AACG induced by topiramate from a case of primary AACG, since they differ in their clinical presentation, mechanism of action, and treatment. Mismanagement can have potentially serious consequences.

Metodología de una revisión sistemática / Methodology of a systematic review

Contexto: La medicina basada en la evidencia tiene como objetivo apoyarse en la mejor información científica disponible para aplicarla a la práctica clínica. Entender e interpretar la evidencia científica implica conocer los niveles de evidencia disponibles, donde las revisiones sistemáticas y/o metaanálisis de ensayos clínicos son la cúspide de la pirámide del nivel de evidencia. Adquisición de la evidencia: El proceso de revisión debe estar bien desarrollado y planificado de antemano para reducir sesgos y eliminar estudios irrelevantes o de baja calidad. Los pasos a seguir para la realización de una revisión sistemática incluyen: (I) formular correctamente la pregunta clínica a responder (PICO), (II) desarrollo de un protocolo (criterios de inclusión y exclusión), (III) realizar una búsqueda bibliográfica detallada y amplia, (IV) cribar los resúmenes de los trabajos identificados en la búsqueda y posteriormente de los textos completos seleccionados (PRISMA). Síntesis de la evidencia: Una vez seleccionados los estudios se debe: (V) extraer en un formulario diseñado en el protocolo los datos necesarios para resumir los estudios incluidos, (VI) evaluar los sesgos de cada estudio pudiendo identificar la calidad de la evidencia disponible y, por último, (VII) desarrollar las tablas y el texto que sinteticen la evidencia. Conclusiones: Una revisión sistemática implica un resumen crítico y reproducible de los resultados de las publicaciones disponibles sobre un mismo tema o pregunta clínica concreta. Con el fin de mejorar la escritura científica, se expone de una forma estructurada la metodología para la realización de una revisión sistemática

Context: The objective of evidence-based medicine is to employ the best scientific information available to apply to clinical practice. Understanding and interpreting the scientific evidence involves understanding the available levels of evidence, where systematic reviews and meta-analyses of clinical trials are at the top of the levels-of-evidence pyramid. Acquisition of evidence: The review process should be well developed and planned to reduce biases and eliminate irrelevant and low-quality studies. The steps for implementing a systematic review include (I) correctly formulating the clinical question to answer (PICO), (II) developing a protocol (inclusion and exclusion criteria), (III) performing a detailed and broad literature search and (IV) screening the abstracts of the studies identified in the search and subsequently of the selected complete texts (PRISMA). Synthesis of the evidence: Once the studies have been selected, we need to (V) extract the necessary data into a form designed in the protocol to summarise the included studies, (VI) assess the biases of each study, identifying the quality of the available evidence, and (VII) develop tables and text that synthesise the evidence. Conclusions: A systematic review involves a critical and reproducible summary of the results of the available publications on a particular topic or clinical question. To improve scientific writing, the methodology is shown in a structured manner to implement a systematic review

Methodology of a systematic review. / Metodología de una revisión sistemática.

CONTEXT: The objective of evidence-based medicine is to employ the best scientific information available to apply to clinical practice. Understanding and interpreting the scientific evidence involves understanding the available levels of evidence, where systematic reviews and meta-analyses of clinical trials are at the top of the levels-of-evidence pyramid. ACQUISITION OF EVIDENCE: The review process should be well developed and planned to reduce biases and eliminate irrelevant and low-quality studies. The steps for implementing a systematic review include (i) correctly formulating the clinical question to answer (PICO), (ii) developing a protocol (inclusion and exclusion criteria), (iii) performing a detailed and broad literature search and (iv) screening the abstracts of the studies identified in the search and subsequently of the selected complete texts (PRISMA). SYNTHESIS OF THE EVIDENCE: Once the studies have been selected, we need to (v) extract the necessary data into a form designed in the protocol to summarise the included studies, (vi) assess the biases of each study, identifying the quality of the available evidence, and (vii) develop tables and text that synthesise the evidence. CONCLUSIONS: A systematic review involves a critical and reproducible summary of the results of the available publications on a particular topic or clinical question. To improve scientific writing, the methodology is shown in a structured manner to implement a systematic review.

Los niveles de plomo en sangre en una muestra representativa de la población adulta española: el proyecto BIOAMBIENT. ES / Blood lead levels in a representative sample of the Spanish adult population: The BIOAMBIENT. ES project

Este trabajo proporciona la primera información de referencia a nivel nacional en relación con la exposición al plomo en la población adulta española. Se midieron los niveles de plomo en sangre en una muestra representativa de la población activa española (1880 sujetos entre 18 y 65 años) con el fin de ayudar a establecer los niveles de referencia, seguir tendencias temporales, identificar a grupos de alta exposición y permitir comparaciones con otros países. Todos los participantes completaron un cuestionario epidemiológico que incluía preguntas sobre sexo, edad, sector de actividad, procedencia geográfica, dieta y estilos de vida. La media geométrica de los niveles de plomo en sangre en la población estudiada fue de 24,0 μg/l (IC95% 23,0-25,1 μg/L), siendo los niveles en las mujeres significativamente menores que en los hombres, 19,5 μg/L (18,5-20,5 μg/L) y 28,3 μg/L (26,7-30,0 μg/L), respectivamente. Los niveles medios de plomo en la sangre fueron más altos en los grupos de más edad para ambos sexos. Las mujeres en edad fértil tuvieron niveles en sangre de 18,0 μg/L (GM). El valor de referencia (percentil 95) de plomo en sangre para el conjunto de la población estudiada fue 56,80 μg/L, siendo de 64,00 μg/L, 44,80 μg/L y 36,00 μg/L para hombres, mujeres y mujeres en edad reproductiva, respectivamente. Los trabajadores del sector de servicios tenían niveles de plomo en sangre inferiores a los de los sectores de construcción, agricultura e industria. Se encontraron pequeñas diferencias geográficas, aunque significativas. En comparación con otros países europeos, la población española estudiada presentaba niveles de plomo similares a los hallados en poblaciones de Francia y Bélgica, y niveles ligeramente inferiores a los de poblaciones de Italia, República Checa, Alemania o el Reino Unido

This paper provides the first baseline information on a national scale regarding lead exposure in the Spanish adult population. Blood lead levels were measured in a representative sample of the Spanish working population (1880 subjects aged 18-65 years) in order to help establish reference levels, follow temporal trends, identify high-exposure groups and to enable comparisons with other countries. All participants completed an epidemiological questionnaire including gender, age, occupational sector, geographic area, and dietary and lifestyle information. We found that the geometric mean of blood lead levels in the study population was 24.0 μg/L (95% CI: 23.0-25.1 μg/L), with women having significantly lower levels than men, 19.5 μg/L (18.5-20.5 μg/L) compared to 28.3 μg/L (26.7-30.0 μg/L), respectively. Mean blood lead levels were higher in elder groups in both genders. Women of a childbearing age had blood levels of18.0 μg/L (GM). Reference values (95%) for lead in blood in the studied population was 56.80 μg/L, with 64.00 μg/L, 44.80 μg/L and 36.00 μg/L for man, women and women of childbearing age, respectively. Workers from the service sector had lower blood lead levels than those from the construction, agricultural and industry sectors. Small, although significant, geographical differences had been found. In an European comparison, the Spanish population studied herein had lead levels similar to populations in countries such as France and Belgium, and slightly lower levels than Italian ,Czech, German or UK populations

Diagnóstico de rabdomioma fetal ecográfico / Ultrasonographic diagnosis of fetal rhabdomyoma

El rabdomioma es una tumoración rara detectada, en la mayor parte de las ocasiones, por ecografía y representa aproximadamente un 1% del total de las anomalías cardíacas halladas intraútero. La evolución intrauterina y su repercusión hemodinámica dependen del crecimiento del tumor. Grandes masas pueden ser causa de obstrucciones y posteriormente de hidropesía y taquiarritmias. A estas complicaciones se añade su asociación a la esclerosis tuberosa, la cual ocupa un lugar importante en el consejo prenatal a los padres, ya que su herencia es autosómica dominante. La frecuencia de la asociación es del 50-70% en las distintas series (AU)

Rhabdomyoma is a rare tumor usually detected by ultrasonography. This neoplasm represents approximately 1% of all intrauterine cardiac abnormalities. Development inside the uterus and the hemodynamic repercussions of this entity depend on tumoral growth. Large masses can cause obstructions and subsequently hydrops and tachyarrhythmias. In addition to these complications, rhabdomyoma is associated with tuberous sclerosis, which occupies an importance place in prenatal counseling since inheritance is autosomal dominant. The frequency of this association is between 50% and 70% in distinct series (AU)

Características clínicas y de personalidad en padres de esquizofrénicos / Clinical and personality characteristics in parents of schizophrenics

Ciento cuarenta parejas casadas, 67 con un hijo con esquizofrenia y 73 controles sin patología, fueron evaluadas con el Eysenck Personality Questionnaire, el Beck Depression Inventory y el Self-Rating Anxiety Scale. Los resultados mostraron puntuaciones más elevadas en ansiedad, depresión, neuroticismo, psicoticismo e inferiores en sinceridad en los padres de esquizofrénicos, frente a los controles. Estos hallazgos sugieren que la ansiedad, depresión y neuroticismo de los padres de esquizofrénicos puede deberse a la carga, pero no el psicoticismo. El tratamiento de la esquizofrenia debería ser complementado con intervenciones dirigidas a la ansiedad de los padres, para que puedan estar en mejores condiciones de ayudar sus hijos

One hundred forty married couples, 67 of them with a child with schizophrenia, and 73 controls without pathology, were assessed with the Eysenck Personality Questionnaire, the Beck Depression Inventory and the Self-Rating Anxiety Scale. Results showed higher scores in anxiety, depression, neuroticism, psychoticism and lower in honesty, in parents of schizophrenics than controls. These findings suggest that the anxiety, depression and neuroticism of the parents with a schizophrenic offspring could be an effect of the burden, but not the psychoticism. Treatment on the schizophrenia should be supplemented by interventions aimed at parents’ anxiety and depression, so that they can be in better conditions to help their child

Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

CDK4 expression and activity are required for cytokine responsiveness in T cells.

Stimulation of lymphocytes through the Ag receptor can lead to cytokine responsiveness or unresponsiveness. We examined the importance of cyclin-dependent kinase (CDK)4 to establish and maintain IL-2 responsiveness in human T cells. Our results show that a herbimycin A- and staurosporine-sensitive phase of CDK4 expression and activity preceded the acquisition of IL-2-responsiveness in mitogen-stimulated peripheral blood T cells. Intriguingly, CDK4 expression and activity were demonstrable in purified unstimulated peripheral blood T cells from approximately 30% (5/16) of healthy individuals examined for this study. These T cells proliferated in response to IL-2 without additional mitogens, and both the expression and activity of CDK4 and the ability to respond to cytokines were resistant to herbimycin A and staurosporine. The pattern of CDK4 expression and response to IL-2 in this subset of individuals resembled that seen in the human IL-2-dependent Kit-225 T cell line. However, in contrast to normal T cells, Kit-225 cells were rendered unresponsive to IL-2 by stimulation through the Ag receptor. In these cells, PHA, anti-CD3, or PMA induced marked reductions of CDK4 expression and activity that paralleled IL-2 unresponsiveness, and these effects were not reversible by IL-2. Furthermore, IL-2-dependent proliferation could be similarly inhibited in Kit-225 cells by overexpression of the CDK inhibitors p16/Ink4-a or p21/Waf-1a or by overexpression of a kinase-inactive CDK4 mutant. The data indicate that CDK4 expression and activity are necessary to induce and maintain cytokine responsiveness in T cells, suggesting that CDK4 is important to link T cell signaling pathways to the machinery that controls cell cycle progression.

[Clinical practice guidelines of the Spanish Society of Cardiology on stable angina]. / Guías de práctica clínica de la Sociedad Española de Cardiología en la angina estable.

Stable angina pectoris is the most common clinical manifestation of chronic ischemic heart disease, the prognosis of which depends on many factors. The authors have analyzed the incidence, evolution and prognosis of this problem in Spain and have reviewed the methods to determine the diagnosis and degree of clinical severity. A careful clinical history, an electrocardiogram, and the evaluation of the severity of the ischemia and left ventricle systolic function are, in most cases, useful to determine the degree of disease severity and establish the prognosis. In other cases, more sophisticated or complex diagnostic techniques such as perfusion tests, stress echocardiography and lastly coronary arteriography, which is the only method currently available to study coronary anatomy are required to carry out an adequate study of the patient. At present, the therapeutic arsenal for the treatment of ischemic coronary disease is extensive, from both a pharmacological and revascularization point of view. Nonetheless it is essential to carry out aggressive therapy to control the risk factors. The decision as to the type of treatment required mainly depends on the severity of the ischemia and in the case of revascularization, on the clinical and anatomical factors as well as on the preferences of the patient and the experience and results of the medical-surgical group involved in the therapy of these patients.

Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition.

We assessed the expression and distribution of p21/Waf-1 in TLM1 melanoma cells that exhibit contact inhibition and require serum for growth. The growth stage of cells stimulated to enter the mitotic cell cycle synchronously and grow to confluence was characterized by distinct, yet consistent levels and patterns of distribution of p21/Waf-1. Significantly, sustained accumulation of p21/Waf-1 in the nuclear compartment was seen only after 4 days in culture when cell-to-cell contacts were established, leading to a diminished rate of cell growth. Overexpression of wild-type waf-1 in melanoma cells reduced growth of subconfluent cells, decreased Cdk4 activity with a concomitant increase in hypophosphorylated Rb, and promoted cell death by apoptosis. The data support the premise that cell-to-cell contacts provide signals that mediate sustained nuclear localization of p21/Waf-1 leading to cell growth arrest; furthermore, an elevation in the activity of this protein can lead to apoptosis.

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

Predicción de afección coronarla a partir de sus factores de riesgo en una población laboral / Prediction of coronary disease from its risk factors in a working population

Hemos llevado a cabo un cálculo de predicción del riesgo coronario a diez años en una población laboral de 1.200 trabajadores de ambos sexos, utilizando la "Tabla de Predicción de Factores de Riesgo de Coronariopatía" basada en el método de análisis multivariante del Estudio de Framinghan. Este procedimiento valora conjuntamente la edad, el sexo, el colesterol total y HDL, la presión arterial sistólica el hábito tabáquico, la existencia de diabetes y la hipertrofia ventricular izquierda. En el colectivo estudiado, el riesgo coronario medio fue de 3,77 ñ 3,82 por ciento en los varones y del 1,53 ñ 1,07 por ciento en las mujeres. El riesgo observado ha sido inferior al teórico esperado en todos los intervalos de edad (AU)

The yeast mitochondrial citrate transport protein. Probing the secondary structure of transmembrane domain iv and identification of residues that likely comprise a portion of the citrate translocation pathway.

The mitochondrial citrate transport protein (CTP) has been investigated by replacing 22 consecutive residues within transmembrane domain IV, one at a time, with cysteine. A cysteine-less CTP retaining wild-type functional properties served as the starting template. The single Cys CTP variants were overexpressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system. The accessibility of each single Cys mutant to three methanethiosulfonate reagents was evaluated by determining the pseudo first order rate constants for inhibition of CTP function. These rate constants varied by seven orders of magnitude. With three independent data sets we observed peaks and troughs in the rate constant data at identical amino acid positions and a periodicity of four was observed from residues 177-193. Based on the pattern of accessibility we conclude that residues 177-193 exist as an alpha-helix. Furthermore, a water-accessible face of the helix has been defined consisting of Pro-177, Val-178, Arg-181, Gln-182, Asn-185, Gln-186, Arg-189, Leu-190, and Tyr-193, and a water-inaccessible face has been delineated consisting of Ser-179, Met-180, Ala-183, Ala-184, Ala-187, Val-188, Gly-191, and Ser-192. We infer that the water-accessible face comprises a portion of the substrate translocation pathway through the CTP, whereas the water-inaccessible surface faces the lipid bilayer.

The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function.

Utilizing site-directed mutagenesis in combination with chemical modification of mutated residues, we have studied the roles of cysteine and arginine residues in the mitochondrial citrate transport protein (CTP) from Saccharomyces cerevisiae. Our strategy consisted of the sequential replacement of each of the four endogenous cysteine residues with Ser or in the case of Cys(73) with Val. Wild-type and mutated forms of the CTP were overexpressed in Escherichia coli, purified, and reconstituted in phospholipid vesicles. During the sequential replacement of each Cys, the effects of both hydrophilic and hydrophobic sulfhydryl reagents were examined. The data indicate that Cys(73) and Cys(256) are primarily responsible for inhibition of the wild-type CTP by hydrophilic sulfhydryl reagents. Experiments conducted with triple Cys replacement mutants (i.e. Cys(192) being the only remaining Cys) indicated that sulfhydryl reagents no longer inhibit but in fact stimulate CTP function 2-3-fold. Following the simultaneous replacement of all four endogenous Cys, the functional properties of the resulting Cys-less CTP were shown to be quite similar to those of the wild-type protein. Finally, utilizing the Cys-less CTP as a template, the roles of Arg(181) and Arg(189), two positively charged residues located within transmembrane domain IV, in CTP function were examined. Replacement of either residue with a Cys abolishes function, whereas replacement with a Lys or a Cys that is subsequently covalently modified with (2-aminoethyl)methanethiosulfonate hydrobromide, a reagent that restores positive charge at this site, supports CTP function. The results clearly show that positive charge at these two positions is essential for CTP function, although the chemistry of the guanidinium residue is not. Finally, these studies: (i) definitely demonstrate that Cys residues do not play an important role in the mechanism of the CTP; (ii) prove the utility of the Cys-less CTP for studying structure/function relationships within this metabolically important protein; and (iii) have led to the hypothesis that the polar face of alpha-helical transmembrane domain IV, within which Arg(181), Arg(189), and Cys(192) are located, constitutes an essential portion of the citrate translocation pathway through the membrane.