RESUMEN
Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.
Asunto(s)
Escherichia coli , Galectina 3 , Isopropil Tiogalactósido , Galectina 3/genética , Galectina 3/metabolismo , Galectina 3/biosíntesis , Galectina 3/química , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Isopropil Tiogalactósido/farmacología , Expresión Génica , Galectinas/genética , Galectinas/metabolismo , Galectinas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismoRESUMEN
BACKGROUND: Phosphate is an essential compound for life. Escherichia coli employs a signal transduction pathway that controls the expression of genes that are required for the high-affinity acquisition of phosphate and the utilization of alternate sources of phosphorous. These genes are only expressed when environmental phosphate is limiting. The seven genes for this signaling pathway encode the two-component regulatory proteins PhoB and PhoR, as well as the high-affinity phosphate transporter PstSCAB and an auxiliary protein called PhoU. As the sensor kinase PhoR has no periplasmic sensory domain, the mechanism by which these cells sense environmental phosphate is not known. This paper explores the hypothesis that it is the alternating conformations of the PstSCAB transporter which are formed as part of the normal phosphate transport cycle that signal phosphate sufficiency or phosphate limitation. RESULTS: We tested two variants of PstB that are predicted to lock the protein in either of two conformations for their signaling output. We observed that the pstBQ160K mutant, predicted to reside in an inward-facing, open conformation signaled phosphate sufficiency whereas the pstBE179Q mutant, predicted to reside in an outward-facing, closed conformation signaled phosphate starvation. Neither mutant showed phosphate transport. CONCLUSIONS: These results support the hypothesis that the alternating conformations of the PstSCAB transporter are sensed by PhoR and PhoU. This sensory mechanism thus controls the alternate autokinase and phospho-PhoB phosphatase activities of PhoR, which ultimately control the signaling state of the response regulator PhoB.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Transducción de Señal/genética , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica/genética , Histidina Quinasa/genética , Histidina Quinasa/metabolismo , Homeostasis/genética , Homeostasis/fisiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Proteínas Quinasas , Alineación de Secuencia , Inanición , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Self-directed treatment has been shown to be effective in the treatment of bulimia nervosa. This paper shows that compliance with a manual is related to outcome. Forty per cent of high compliers achieved full remission after eight weeks with a manual, compared with only 5 per cent of low compliers. Furthermore, patients with greater weight and shape concerns were less likely to use a manual while those with a longer duration of illness were more likely to use it.
Asunto(s)
Bulimia/terapia , Cooperación del Paciente , Psicoterapia/métodos , Autocuidado/métodos , Autocuidado/normas , Adulto , Imagen Corporal , Bulimia/diagnóstico , Bulimia/psicología , Distribución de Chi-Cuadrado , Estudios de Evaluación como Asunto , Femenino , Estudios de Seguimiento , Humanos , Cooperación del Paciente/psicología , Pronóstico , Análisis de Regresión , Estrés Psicológico , Factores de TiempoRESUMEN
The measurement of health-related quality of life has attracted much attention and is now a component of most big clinical trials. As we evaluate and choose between treatments and make judgements about priorities in health care, it is important that the equivalent data are available for different patient groups.
Asunto(s)
Anorexia Nerviosa/psicología , Bulimia/psicología , Calidad de Vida , Actividades Cotidianas , Adolescente , Adulto , Enfermedad Coronaria/psicología , Fibrosis Quística/psicología , Emociones , Femenino , Trasplante de Corazón/psicología , Humanos , Relaciones Interpersonales , Masculino , Trastornos del Sueño-Vigilia , Aislamiento Social , Movilidad SocialRESUMEN
OBJECTIVE: To test the short term efficacy of a self directed treatment manual for bulimia nervosa. DESIGN: Randomised controlled trial of the manual against cognitive behavioural therapy and a waiting list. SETTING: Tertiary referral centre. SUBJECTS: 81 consecutive referrals presenting with bulimia nervosa or atypical bulimia nervosa. MAIN OUTCOME MEASURES: Frequency of binge eating, vomiting, and other behaviours to control weight as well as abstinence from these behaviours. RESULTS: Cognitive behavioural treatment produced a significant reduction in the frequency of binge eating, vomiting, and other behaviours to control weight. The manual significantly reduced frequency of binge eating and weight control behaviours other than vomiting, and there was no change in the group on the waiting list. Full remission was achieved in five (24%) of the group assigned to cognitive behavioural treatment, nine (22%) of the group who used the manual, and two (11%) of the group on the waiting list. CONCLUSIONS: A self directed treatment manual may be a useful first intervention in the treatment of bulimia nervosa.