Efectos de la rotigotina sobre la sintomatología, calidad de vida e higiene de sueño en el síndrome de piernas inquietas en hemodiálisis / Effects of rotigotine on clinical symptoms, quality of life and sleep hygiene adequacy in haemodialysis-associated restless legs syndrome

Introducción: El síndrome de piernas inquietas (SPI) es un trastorno neurológico caracterizado por una molesta sintomatología, asociado a deterioro de calidad de vida e higiene de sueño. Rotigotina constituye una novedosa alternativa terapéutica, si bien existen escasos estudios publicados sobre rotigotina en pacientes en hemodiálisis (HD) con SPI. Objetivos: 1.- Establecer la prevalencia de SPI en nuestra unidad de HD. 2.- Evaluar la eficacia y el perfil de seguridad asociado a rotigotina así como su efecto sobre la sintomatología, calidad de vida e higiene del sueño en nuestra población en HD con SPI. Material y métodos: Estudio unicéntrico, prospectivo de 12 semanas de duración. Dos fases (6 semanas): fase 1 (no tratamiento) y fase 2 (rotigotina). Analizamos: 1.- Datos demográficos, bioquímicos, parámetros de adecuación de HD y tratamiento médico relacionado con SPI. 2.- Cuestionario sobre síntomas en extremidades inferiores (QS). 3.- Escala de gravedad de los síntomas (GRLS). 4.- Calidad de vida SPI: John Hopkins RLS-QoL (JH-QoL). 5.- Higiene del sueño: Escala SCOPA. Resultados: Se incluyó a 66 pacientes en HD. De ellos, 14 con SPI; el 44,4% eran hombres, con 70,2±9,9 años y 111,1±160,8 meses en HD. El 22,9%, con SPI. Únicamente en la fase 2 observamos una mejoría significativa para QS (10±2,4 vs. 5,7±1,0), GRLS (21±4 vs. 5,7±4,6), JH-QoL (22,1±4,4 vs. 4,3±4,0) y SCOPA (16±5,3 vs. 6,7±1,9). Un 77,7 y un 11,1% presentaron remisión parcial (>20%) y completa (>80%), respectivamente. Un 55,5% alcanzó sintomatología «cero». Un único paciente presentó intolerancia digestiva y ninguno, augmentation efect. No observamos cambios en datos bioquímicos, adecuación dialítica ni tratamiento médico. El análisis intergrupos mostró una mejoría significativa en la fase 2 con relación a QS, GRLSS, JH-QoL y SCOPA. Conclusiones: En nuestro estudio, el SPI urémico presentó una prevalencia considerable. Rotigotina mejoró la sintomatología clínica, la calidad de vida y la higiene de sueño en los pacientes con SPI en HD, por lo que resulta ser un fármaco seguro, con mínimos efectos adversos y con cumplimento terapéutico completo. No obstante, serían necesarios futuros estudios para confirmar el beneficio de rotigotina en la población en HD con SPI (AU)

Background: Restless legs syndrome (RLS) is a neurological disorder characterised by bothersome symptoms associated with impaired quality of life and sleep hygiene. Rotigotine is a novel therapeutic alternative, although few studies have been published in patients on haemodialysis (HD) with RLS treated with rotigotine. Objectives: 1.- To establish the prevalence of RLS in our HD unit. 2.- To evaluate the efficacy and safety profile of rotigotine and its effect on symptoms, quality of life and sleep hygiene in our HD population with RLS. Material and methods: A single-centre, 12-week prospective study. Two stages (6 weeks): stage 1 (no treatment) and stage 2 (rotigotine). We analysed: 1.- Demographic data, biochemistry data, HD suitability parameters and RLS medical treatment data. 2.- Lower extremity symptoms questionnaire (QS). 3.- RLS severity symptoms scale (SRLSS). 4.- RLS Quality of life: John Hopkins RLS-QoL (JH-QoL). 5.- Sleep hygiene: SCOPA Scale. Results: We included 66 HD patients, 14 with RLS; 44.4% male, 70.2±9.9 years and 111.1±160.8 months on HD. And 22.9% RLS. Exclusively in stage 2, a significant improvement for QS (10±2.4 vs. 5.7±1.0), SRLSS (21±4 vs. 5.7±4.6), JH-QoL (22.1±4.4 vs. 4.3±4.0) and SCOPA (16±5.3 vs. 6.7±1.9) were observed. A 77.7 and 11.1%, showed partial (> 20%) and complete (> 80%) remission, respectively, while 55.5% achieved «zero» symptoms. Only one patient had gastrointestinal intolerance and none experienced augmentation effect. No changes in biochemical data, suitability for dialysis or medical treatment were found. The inter-group analysis showed a significant improvement in relation to QS, SRLSS, JH-QoL and SCOPA in stage 2. Conclusions: RLS showed a considerable prevalence in our HD unit. Rotigotine improved clinical symptoms, quality of life and sleep hygiene in RLS patients on HD and was found to be a safe drug with minimal side effects and total therapeutic compliance. Nevertheless, future studies should be performed to confirm the benefits of rotigotine in RLS patients on haemodialysis (AU)

Effects of rotigotine on clinical symptoms, quality of life and sleep hygiene adequacy in haemodialysis-associated restless legs syndrome. / Efectos de la rotigotina sobre la sintomatología, calidad de vida e higiene de sueño en el síndrome de piernas inquietas en hemodiálisis.

BACKGROUND: Restless legs syndrome (RLS) is a neurological disorder characterised by bothersome symptoms associated with impaired quality of life and sleep hygiene. Rotigotine is a novel therapeutic alternative, although few studies have been published in patients on haemodialysis (HD) with RLS treated with rotigotine. OBJECTIVES: 1.- To establish the prevalence of RLS in our HD unit. 2.- To evaluate the efficacy and safety profile of rotigotine and its effect on symptoms, quality of life and sleep hygiene in our HD population with RLS. MATERIAL AND METHODS: A single-centre, 12-week prospective study. Two stages (6 weeks): stage 1 (no treatment) and stage 2 (rotigotine). We analysed: 1.- Demographic data, biochemistry data, HD suitability parameters and RLS medical treatment data. 2.- Lower extremity symptoms questionnaire (QS). 3.- RLS severity symptoms scale (SRLSS). 4.- RLS Quality of life: John Hopkins RLS-QoL (JH-QoL). 5.- Sleep hygiene: SCOPA Scale. RESULTS: We included 66 HD patients, 14 with RLS; 44.4% male, 70.2±9.9 years and 111.1±160.8 months on HD. And 22.9% RLS. Exclusively in stage 2, a significant improvement for QS (10±2.4 vs. 5.7±1.0), SRLSS (21±4 vs. 5.7±4.6), JH-QoL (22.1±4.4 vs. 4.3±4.0) and SCOPA (16±5.3 vs. 6.7±1.9) were observed. A 77.7 and 11.1%, showed partial (> 20%) and complete (> 80%) remission, respectively, while 55.5% achieved «zero¼ symptoms. Only one patient had gastrointestinal intolerance and none experienced augmentation effect. No changes in biochemical data, suitability for dialysis or medical treatment were found. The inter-group analysis showed a significant improvement in relation to QS, SRLSS, JH-QoL and SCOPA in stage 2. CONCLUSIONS: RLS showed a considerable prevalence in our HD unit. Rotigotine improved clinical symptoms, quality of life and sleep hygiene in RLS patients on HD and was found to be a safe drug with minimal side effects and total therapeutic compliance. Nevertheless, future studies should be performed to confirm the benefits of rotigotine in RLS patients on haemodialysis.

Neuromuscular electrostimulation: a new therapeutic option to improve radio-cephalic arteriovenous fistula maturation in end-stage chronic kidney disease patients.

BACKGROUND: Radio-cephalic arteriovenous fistula (RCAVF) is the gold standard vascular access for end-stage chronic kidney disease patients. Exercises after arteriovenous fistula (AVF) creation improve maturation. No articles are published regarding neuromuscular electrostimulation (NMES) in AVF maturation. OBJECTIVES: To assess the usefulness of a NMES programme on RCAVF maturation process. METHODS: An 8-week single-centre prospective study. Two groups were established: control group (CG): underwent usual RCAVF forearm exercises and electrostimulation group (ESG): underwent RCAVF NMES programme. Handgrip (HG) measurement, preoperative Doppler ultrasonography (DUS) parameters, clinical and DUS maturation as well as surgical complications were assessed. RESULTS: Thirty-six patients (54% men). Mean age 67.9 ± 14.3 years; 12 ESG and 24 CG. Demographic data, comorbidities, medical treatment, HG and DUS measurement at baseline were similar. HG increased in both groups at the end of the study (CG 24.5 ± 9.5 vs. 26.1 ± 10.1 kg, p 0.048; ESG 25.8 ± 10.3 vs. 26.3 ± 11.6 kg, p 0.644). RCAVF forearm vein diameter (CG 3.1 ± 0.7 vs. 5.7 ± 1.1 mm; ESG 2.9 ± 0.8 vs. 6.1 ± 1.7 mm) and humeral artery blood flow rate (CG 110.5 ± 20.7 vs. 1053.4 ± 510.7 ml/min; ESG 118.2 ± 31.6 vs. 954.1 ± 542.2 ml/min) statistically increased for both groups. A significant increase in clinical maturation in ESG (62.5 vs. 91.7%, p 0.046) at 8 weeks was observed. Four patients in each group developed juxta-anastomotic stenosis and were surgically repaired. No adverse NMES effects were registered. CONCLUSIONS: NMES of forearm muscles is a safe and effective technique to improve RCAVF maturation and constitutes a novel alternative to forearm isometrics exercises. Nevertheless, further studies are required to confirm the potential effect of NMES in the vascular access maturation process.

Clinical Utility of a New Predicting Score for Radiocephalic Arteriovenous Fistula Survival.

BACKGROUND: Radiocephalic arteriovenous fistula (RC-AVF) is the recommended first choice for vascular access (VA). The CAVeA2T2 scoring system was recently published (ipsilateral central venous catheter access, age >73 years, vein <2.2 mm, lower limb angioplasty, and absent intraoperative thrill). The aim of the present study was to assess the clinical utility of the CAVeA2T2 scoring system for predicting RC-AVFs survival in our center and its subsequent application in VA management. MATERIAL AND METHODS: In this single-center retrospective study, all RC-AVFs performed from January 2010 to July 2014 were included. The CAVeA2T2 was applied. Primary, assisted primary, and secondary patency rates were measured. RESULTS: Sixty RC-AVFs were analyzed. Mean age was 64.3 ± 14.7 years. Mean CAVeA2T2 score was 1.23 ± 1.2. The median fistula secondary patency was 13.7 ± 1.6 months. Secondary patency was at 6 weeks and at 6, 12, and 24 months: 88.3%, 66.7%, 55%, and 31.7%, respectively. Increasing score (≥2) was associated with a decrease in primary (log-rank, χ2 = 16.7, dif = 1, P = 0.0001) and secondary patency rate survival (log-rank, χ2 = 5.4, dif = 1, P = 0.0001). In addition, stratification of the CAVeA2T2 score into 3 groups (scores 0-1, 2, and 3+) retained its significance for primary (log-rank, χ2 = 19.4, dif = 2, P = 0.0001) and secondary patency rate survival (log-rank, χ2 = 5.5, dif = 2, P = 0.046) at the end of the study. CONCLUSIONS: In the present study, the CAVeA2T2 scoring system has proved to be a useful, easy to apply tool that is highly predictive of RC-AVF survival. Based on our results, we should avoid perform RC-AVFs, in those patients with CAVeA2T2 score ≥2 and late nephrology referral. Prospective studies should be designed to establish the management of patients with a higher CAVeA2T2 score.

Efecto de la electroestimulación neuromuscular sobre la fuerza muscular, capacidad funcional y composición corporal en los pacientes en hemodiálisis / The effect of neuromuscular electrical stimulation on muscle strength, functional capacity and body composition in haemodialysis patients

Introducción: Los pacientes en hemodiálisis (HD) se caracterizan por una gran pérdida muscular. Recientemente, la electroestimulación neuromuscular (EENM) constituye una nueva alternativa terapéutica para mejorar la condición física de estos pacientes. No existen estudios acerca de la EENM sobre la composición corporal en HD. Objetivo: Analizar el efecto de la EENM sobre la fuerza muscular, capacidad funcional y composición corporal en nuestros pacientes en HD. Material y métodos: Estudio prospectivo unicéntrico (12 semanas). Los pacientes fueron asignados a grupo electroestimulación (EM) o control (CO). El grupo EM incluía un programa de electroestimulación cuadricipital intradiálisis (Compex® Theta 500i). El grupo C recibió cuidado habitual en HD. Analizamos: 1) parámetros nutricionales; 2) composición muscular del cuádriceps; 3) fuerza de extensión máxima del cuádriceps (FEMQ) y handgrip (HG); 4) sit to stand to sit (STS10), six-minutes walking test (6MWT) y 5) composición corporal (bioimpedancia eléctrica). Resultados: De un total de 20 pacientes, el 55% fueron hombres. Edad media: 67,7 años, con 30,3 meses en HD. Principal etiología: DM (35%). Hubo 13 pacientes en EM y 7 en el grupo CO. Al final del estudio, únicamente EM presentó mejoría en (*p<0,05): FEMQ* (11,7±7,1 vs. 13,4±7,4kg), STS10 (39,3±15,5 vs. 35,8±13,7s) y 6MWT* (9,9%; 293,2 vs. 325,2m). Igualmente, el grupo EM incrementó el área muscular (AMQ*: 128,6±30,2 vs. 144,6±22,4cm2) y disminuyó el área grasa cuadricipital (AGQ*: 76,5±26,9 vs. 62,1±20,1cm2). No se observaron cambios relevantes en el resto de la composición corporal, parámetros nutricionales ni adecuación dialítica. Conclusiones: 1) La EENM mejoró la fuerza muscular, la capacidad funcional y la composición muscular del cuádriceps de nuestros pacientes. 2) Con los resultados obtenidos, la EENM podría ser una nueva alternativa terapéutica para evitar la atrofia muscular y el deterioro progresivo de la condición física de estos pacientes. 3) No obstante, serían necesarios futuros estudios para establecer los potenciales efectos beneficiosos de la EENM en los pacientes en HD (AU)

Introduction: Haemodialysis (HD) patients are characterised by significant muscle loss. Recently, neuromuscular electrical stimulation (NMES) has emerged as a new therapeutic alternative to improve these patients’ physical condition. To date, no studies on the effects of NMES on body composition in HD patients have been published. Objective: To analyse the effect of NMES on muscle strength, functional capacity and body composition in our HD patients. Material and methods: A 12-week, single-centre, prospective study. The patients were assigned to an electrical stimulation (ES) or control (CO) group. The ES group was subjected to intradialytic electrical stimulation of the quadriceps (Compex® Theta 500i), while the CO group received standard HD care. We analysed the following: 1) nutritional parameters; 2) muscle composition of the quadriceps; 3) maximum quadriceps extension strength (mes) and hand-grip (HG); 4) «sit to stand to sit» (STS10) and «six-minute walking test» (6MWT); 5) body composition (bioelectrical impedance analysis). Results: Of 20 patients, 55% were men. Mean age 67.7 years, 30.3 months in HD. Main aetiology: DM (35%). In the ES group were 13 patients, and 7 in the CO group. At the end of the study, an improvement was only observed in the ES group (*P<.05): MES* (11.7±7.1 vs. 13.4±7.4kg), STS10 (39.3±15.5 vs. 35.8±13.7s) and 6MWT* (9.9%, 293.2 vs. 325.2m). Furthermore, increased quadriceps muscle area (QMA*: 128.6±30.2 vs. 144.6±22.4cm2) and lowered quadriceps fat area (QFA*: 76.5±26.9 vs. 62.1±20.1cm2) were observed. No relevant changes in body composition, nutritional parameters and dialysis adequacy were found. Conclusions: 1) NMES improved muscle strength, functional capacity and quadriceps muscle composition in our patients. 2) Based on the results obtained, NMES could be a new therapeutic alternative to prevent muscle atrophy and progressive physical deterioration. 3) However, future studies are necessary to establish the potential beneficial effects of NMES in HD patients (AU)

The effect of neuromuscular electrical stimulation on muscle strength, functional capacity and body composition in haemodialysis patients. / Efecto de la electroestimulación neuromuscular sobre la fuerza muscular, capacidad funcional y composición corporal en los pacientes en hemodiálisis.

INTRODUCTION: Haemodialysis (HD) patients are characterised by significant muscle loss. Recently, neuromuscular electrical stimulation (NMES) has emerged as a new therapeutic alternative to improve these patients' physical condition. To date, no studies on the effects of NMES on body composition in HD patients have been published. OBJECTIVE: To analyse the effect of NMES on muscle strength, functional capacity and body composition in our HD patients. MATERIAL AND METHODS: A 12-week, single-centre, prospective study. The patients were assigned to an electrical stimulation (ES) or control (CO) group. The ES group was subjected to intradialytic electrical stimulation of the quadriceps (Compex® Theta 500i), while the CO group received standard HD care. We analysed the following: 1) nutritional parameters; 2) muscle composition of the quadriceps; 3) maximum quadriceps extension strength (mes) and hand-grip (HG); 4) «sit to stand to sit¼ (STS10) and «six-minute walking test¼ (6MWT); 5) body composition (bioelectrical impedance analysis). RESULTS: Of 20 patients, 55% were men. Mean age 67.7 years, 30.3 months in HD. Main aetiology: DM (35%). In the ES group were 13 patients, and 7 in the CO group. At the end of the study, an improvement was only observed in the ES group (*P<.05): MES* (11.7±7.1 vs. 13.4±7.4kg), STS10 (39.3±15.5 vs. 35.8±13.7s) and 6MWT* (9.9%, 293.2 vs. 325.2m). Furthermore, increased quadriceps muscle area (QMA*: 128.6±30.2 vs. 144.6±22.4cm2) and lowered quadriceps fat area (QFA*: 76.5±26.9 vs. 62.1±20.1cm2) were observed. No relevant changes in body composition, nutritional parameters and dialysis adequacy were found. CONCLUSIONS: 1) NMES improved muscle strength, functional capacity and quadriceps muscle composition in our patients. 2) Based on the results obtained, NMES could be a new therapeutic alternative to prevent muscle atrophy and progressive physical deterioration. 3) However, future studies are necessary to establish the potential beneficial effects of NMES in HD patients.

Magnetic resonance spectroscopy and imaging on fresh human brain tumor biopsies at microscopic resolution.

The metabolic composition and concentration knowledge provided by magnetic resonance spectroscopy (MRS) liquid and high-resolution magic angle spinning spectroscopy (HR-MAS) has a relevant impact in clinical practice during magnetic resonance imaging (MRI) monitoring of human tumors. In addition, the combination of morphological and chemical information by MRI and MRS has been particularly useful for diagnosis and prognosis of tumor evolution. MRI spatial resolution reachable in human beings is limited for safety reasons and the demanding necessary conditions are only applicable on experimental model animals. Nevertheless, MRS and MRI can be performed on human biopsies at high spatial resolution, enough to allow a direct correlation between the chemical information and the histological features observed in such biopsies. Although HR-MAS is nowadays a well-established technique for spectroscopic analysis of tumor biopsies, with this approach just a mean metabolic profile of the whole sample can be obtained and thus the high histological heterogeneity of some important tumors is mostly neglected. The value of metabolic HR-MAS data strongly depends on a wide statistical analysis and usually the microanatomical rationale for the correlation between histology and spectroscopy is lost. We present here a different approach for the combined use of MRI and MRS on fresh human brain tumor biopsies with native contrast. This approach has been designed to achieve high spatial (18 × 18 × 50 µm) and spectral (0.031 µL) resolution in order to obtain as much spatially detailed morphological and metabolical information as possible without any previous treatment that can alter the sample. The preservation of native tissue conditions can provide information that can be translated to in vivo studies and additionally opens the possibility of performing other techniques to obtain complementary information from the same sample.

Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB).

The eukaryotic polypyrimidine tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposite sides that face away from each other. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed ß-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.

Development of a three-dimensional cell culture system based on microfluidics for nuclear magnetic resonance and optical monitoring.

A new microfluidic cell culture device compatible with real-time nuclear magnetic resonance (NMR) is presented here. The intended application is the long-term monitoring of 3D cell cultures by several techniques. The system has been designed to fit inside commercially available NMR equipment to obtain maximum readout resolution when working with small samples. Moreover, the microfluidic device integrates a fibre-optic-based sensor to monitor parameters such as oxygen, pH, or temperature during NMR monitoring, and it also allows the use of optical microscopy techniques such as confocal fluorescence microscopy. This manuscript reports the initial trials culturing neurospheres inside the microchamber of this device and the preliminary images and spatially localised spectra obtained by NMR. The images show the presence of a necrotic area in the interior of the neurospheres, as is frequently observed in histological preparations; this phenomenon appears whenever the distance between the cells and fresh nutrients impairs the diffusion of oxygen. Moreover, the spectra acquired in a volume of 8 nl inside the neurosphere show an accumulation of lactate and lipids, which are indicative of anoxic conditions. Additionally, a basis for general temperature control and monitoring and a graphical control software have been developed and are also described. The complete platform will allow biomedical assays of therapeutic agents to be performed in the early phases of therapeutic development. Thus, small quantities of drugs or advanced nanodevices may be studied long-term under simulated living conditions that mimic the flow and distribution of nutrients.

Use of 1H and 31P HRMAS to evaluate the relationship between quantitative alterations in metabolite concentrations and tissue features in human brain tumour biopsies.

Quantitative multinuclear high-resolution magic angle spinning was performed in order to determine the tissue pH values of and the absolute metabolite concentrations in 33 samples of human brain tumour tissue. Metabolite concentrations were quantified by 1D (1)H and (31)P HRMAS using the electronic reference to in vivo concentrations (ERETIC) synthetic signal. (1)H-(1)H homonuclear and (1)H-(31)P heteronuclear correlation experiments enabled the direct assessment of the (1)H-(31)P spin systems for signals that suffered from overlapping in the 1D (1)H spectra, and linked the information present in the 1D (1)H and (31)P spectra. Afterwards, the main histological features were determined, and high heterogeneity in the tumour content, necrotic content and nonaffected tissue content was observed. The metabolite profiles obtained by HRMAS showed characteristics typical of tumour tissues: rather low levels of energetic molecules and increased concentrations of protective metabolites. Nevertheless, these characteristics were more strongly correlated with the total amount of living tissue than with the tumour cell contents of the samples alone, which could indicate that the sampling conditions make a significant contribution aside from the effect of tumour development in vivo. The use of methylene diphosphonic acid as a chemical shift and concentration reference for the (31)P HRMAS spectra of tissues presented important drawbacks due to its interaction with the tissue. Moreover, the pH data obtained from (31)P HRMAS enabled us to establish a correlation between the pH and the distance between the N(CH(3))(3) signals of phosphocholine and choline in (1)H spectra of the tissue in these tumour samples.

Magnetic resonance microscopy contribution to interpret high-resolution magic angle spinning metabolomic data of human tumor tissue.

HRMAS NMR is considered a valuable technique to obtain detailed metabolic profile of unprocessed tissues. To properly interpret the HRMAS metabolomic results, detailed information of the actual state of the sample inside the rotor is needed. MRM (Magnetic Resonance Microscopy) was applied for obtaining structural and spatially localized metabolic information of the samples inside the HRMAS rotors. The tissue was observed stuck to the rotor wall under the effect of HRMAS spinning. MRM spectroscopy showed a transference of metabolites from the tissue to the medium. The sample shape and the metabolite transfer after HRMAS indicated that tissue had undergone alterations and it can not be strictly considered as intact. This must be considered when HRMAS is used for metabolic tissue characterization, and it is expected to be highly dependent on the manipulation of the sample. The localized spectroscopic information of MRM reveals the biochemical compartmentalization on tissue samples hidden in the HRMAS spectrum.

NMR metabolic profile of human follicular fluid.

The environment of the oocyte during its in vivo maturation consists of follicular fluid (FF) and is surrounded by granulosa cells. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall, that contain a large variety of growth factors, cytokines, amino acids, and other metabolites. These metabolites are presumably involved in the physiology of the oocyte. The identification, quantification and study of FF metabolites can provide additional information about the oocyte state which can be helpful in distinguishing those oocytes that have a greater capacity to be fertilised and to develop properly. The aim of this work is to identify the metabolic profile of FF samples exhaustively using High Resolution Nuclear Magnetic Resonance (NMR). A total of 30 FF samples from oocyte donors (<35 years) were analysed. Different monodimensional (1D) and bidimensional (2D) (homo and heteronuclear) NMR experiments were acquired. A total of 131 chemical shifts were assigned and 42 metabolites, including as example glucose, lactate, acetate, acetoacetate, pyruvate and beta-hydroxybutyrate, were identified. High correlations were found between these important intermediaries of the energetic metabolic pathways of the follicle which can indicate the importance of these pathways in oocyte development. Some of these identified metabolites might be useful as biomarkers of the follicular maturation state, allowing oocytes with a higher fertilisation potential to be selected, thereby increasing pregnancy rates in women following in vitro fertilisation (IVF) treatments.

Structural insights into the GTPase domain of Escherichia coli MnmE protein.

The Escherichia coli MnmE protein is a 50-kDa multidomain GTPase involved in tRNA modification. Its homologues in eukaryotes are crucial for mitochondrial respiration and, thus, it is thought that the human protein might be involved in mitochondrial diseases. Unlike Ras, MnmE shows a high intrinsic GTPase activity and requires effective GTP hydrolysis, and not simply GTP binding, to be functionally active. The isolated MnmE G-domain (165 residues) conserves the GTPase activity of the entire protein, suggesting that it contains the catalytic residues for GTP hydrolysis. To explore the GTP hydrolysis mechanism of MnmE, we analyzed the effect of low pH on binding and hydrolysis of GTP, as well as on the formation of a MnmE transition state mimic. GTP hydrolysis by MnmE, but not GTP binding or formation of a complex with mant-GDP and aluminium fluoride, is impaired at acidic pH, suggesting that the chemistry of the transition state mimic is different to that of the true transition state, and that some residue(s), critical for GTP hydrolysis, is severely affected by low pH. We use a nuclear magnetic resonance (NMR)-based approach to get insights into the MnmE structure and properties. The combined use of NMR restraints and homology structural information allowed the determination of the MnmE G-domain structure in its free form. Chemical shift structure-based prediction provided a good basis for structure refinement and validation. Our data support that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis, although Arg252 may play a role in stabilization of the transition state.

Snake venom disintegrins: evolution of structure and function.

Disintegrins represent a family of polypeptides present in the venoms of various vipers that selectively block the function of integrin receptors. Here, we review our current view and hypothesis on the emergence and the structural and functional diversification of disintegrins by accelerated evolution and the selective loss of disulfide bonds of duplicated genes. Research on disintegrins is relevant for understanding the biology of viper venom toxins, but also provides information on new structural determinants involved in integrin recognition that may be useful in basic and clinical research. The role of the composition, conformation, and dynamics of the integrin inhibitory loop acting in concert with the C-terminal tail in determining the selective inhibition of integrin receptors is discussed.

Conformation and concerted dynamics of the integrin-binding site and the C-terminal region of echistatin revealed by homonuclear NMR.

Echistatin is a potent antagonist of the integrins alpha(v)beta3, alpha5beta1 and alpha(IIb)beta3. Its full inhibitory activity depends on an RGD (Arg-Gly-Asp) motif expressed at the tip of the integrin-binding loop and on its C-terminal tail. Previous NMR structures of echistatin showed a poorly defined integrin-recognition sequence and an incomplete C-terminal tail, which left the molecular basis of the functional synergy between the RGD loop and the C-terminal region unresolved. We report a high-resolution structure of echistatin and an analysis of its internal motions by off-resonance ROESY (rotating-frame Overhauser enhancement spectroscopy). The full-length C-terminal polypeptide is visible as a beta-hairpin running parallel to the RGD loop and exposing at the tip residues Pro43, His44 and Lys45. The side chains of the amino acids of the RGD motif have well-defined conformations. The integrin-binding loop displays an overall movement with maximal amplitude of 30 degrees . Internal angular motions in the 100-300 ps timescale indicate increased flexibility for the backbone atoms at the base of the integrin-recognition loop. In addition, backbone atoms of the amino acids Ala23 (flanking the R24GD26 tripeptide) and Asp26 of the integrin-binding motif showed increased angular mobility, suggesting the existence of major and minor hinge effects at the base and the tip, respectively, of the RGD loop. A strong network of NOEs (nuclear Overhauser effects) between residues of the RGD loop and the C-terminal tail indicate concerted motions between these two functional regions. A full-length echistatin-alpha(v)beta3 docking model suggests that echistatin's C-terminal amino acids may contact alpha(v)-subunit residues and provides new insights to delineate structure-function correlations.

Ionic self-complementarity induces amyloid-like fibril formation in an isolated domain of a plant copper metallochaperone protein.

BACKGROUND: Arabidopsis thaliana copper metallochaperone CCH is a functional homologue of yeast antioxidant ATX1, involved in cytosolic copper transport. In higher plants, CCH has to be transported to specialised cells through plasmodesmata, being the only metallochaperone reported to date that leaves the cell where it is synthesised. CCH has two different domains, the N-terminal domain conserved among other copper-metallochaperones and a C-terminal domain absent in all the identified non-plant metallochaperones. The aim of the present study was the biochemical and biophysical characterisation of the C-terminal domain of the copper metallochaperone CCH. RESULTS: The conformational behaviour of the isolated C-domain in solution is complex and implies the adoption of mixed conformations in different environments. The ionic self-complementary peptide KTEAETKTEAKVDAKADVE, derived from the C-domain of CCH, adopts and extended conformation in solution with a high content in beta-sheet structure that induces a pH-dependent fibril formation. Freeze drying electron microscopy studies revealed the existence of well ordered amyloid-like fibrils in preparations from both the C-domain and its derivative peptide. CONCLUSION: A number of proteins related with copper homeostasis have a high tendency to form fibrils. The determinants for fibril formation, as well as the possible physiological role are not fully understood. Here we show that the plant exclusive C-domain of the copper metallochaperone CCH has conformational plasticity and forms fibrils at defined experimental conditions. The putative influence of these properties with plant copper delivery will be addressed in the future.

The structural plasticity of the C terminus of p21Cip1 is a determinant for target protein recognition.

The cyclin-dependent kinase inhibitory protein p21(Cip1) might play multiple roles in cell-cycle regulation through interaction of its C-terminal domain with a defined set of cellular proteins such as proliferating cell nuclear antigen (PCNA), calmodulin (CaM), and the oncoprotein SET. p21(Cip1) could be described as an intrinsically unstructured protein in solution although the C-terminal domain adopts a well-defined extended conformation when bound to PCNA. However, the molecular mechanism of the interaction with CaM and the oncoprotein SET is not well understood, partly because of the lack of structural information. In this work, a peptide derived from the C-terminal domain of p21(Cip1) that covers the binding domain of the three above-mentioned proteins was used to demonstrate that the C-terminal domain of p21 recognizes multiple ligands through its ability to adopt multiple conformations. The conformation is dictated by tertiary contacts rather than by the primary sequence of the protein. Our results suggest that the C-terminal domain of p21(Cip1) adopts an extended structure when bound to PCNA and probably when bound to the oncoprotein SET, but an alpha helix when bound to CaM.