Evidence for effect of l-serine, a novel therapy for GRIN2B-related neurodevelopmental disorder.

RATIONALE: To date, causal therapy is potentially available for GRIN2B-related neurodevelopmental disorder (NDD) due to loss-of-function (LoF) variants in GRIN2B, resulting in dysfunction of the GluN2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). Recently, in vitro experiments showed that high doses of NMDAR co-agonist d-serine has the potential to boost the activity in GluN2B LoF variant-containing NMDARs. Initial reports of GRIN2B-NDD patients LoF variants, treated with l-serine using different regimens, showed varying effects on motor and cognitive performance, communication, behavior and EEG. Here, this novel treatment using a standardized protocol with an innovative developmental outcome measure is explored further in an open-label observational GRIN2B-NDD study. METHODS: Initially, in vitro studies were conducted in order to functionally stratify two de novo GRIN2B variants present in two female patients (18 months and 4 years old). Functional studies showed that both variants are LoF, and thus the patients were treated experimentally according to an approved protocol with oral l-serine (500 mg/kg/day in 4 doses) for a period of 12 months. Both patients showed a heterogeneous clinical phenotype, however overlapping symptoms were present: intellectual developmental disability (IDD), behavioral abnormalities and hypotonia. Outcome measures included laboratory tests, quality of life, sleep, irritability, stool, and performance skills, measured by, among others, the Perceive-Recall-Plan-Perform System of Task Analysis (PRPP-Assessment). RESULTS: Both patients tolerated l-serine without adverse effects. In one patient, improvement in psychomotor development and cognitive functioning was observed after 12 months (PRPP mastery score 10% at baseline, 78% at twelve months). In the most severe clinically affected patient no significant objective improvement in validated outcomes was observed. Caregivers of both patients reported subjective increase of alertness and improved communication skills. CONCLUSION: Our observational study confirms that l-serine supplementation is safe in patients with GRIN2B-NDD associated with LoF variants, and may accelerate psychomotor development and ameliorate cognitive performance in some but not all patients. The PRPP-Assessment, a promising instrument to evaluate everyday activities and enhance personalized and value-based care, was not performed in the severely affected patient, meaning that possible positive results may have been missed. To generate stronger evidence for effect of l-serine in GRIN2B-NDD, we will perform placebo-controlled n-of-1 trials.

Binding interactions between suberin monomer components and pesticides.

Understanding the role of biomacromolecules and their interactions with pollutants is a key for elucidating the sorption mechanisms and making an accurate assessment of the environmental fate of pollutants. The knowledge of the sorption properties of the different constituents of these biomacromolecules may furnish a significant contribution to this purpose. Suberin is a very abundant biopolymer in higher plants. In this study, suberin monomers isolated from cork were analyzed by thermally-assisted methylation with tetramethylammonium hydroxide (TMAH) in a pyrolysis unit coupled to gas chromatography-mass spectrometry (GC/MS). The isolated monomer mixture was used to study the sorption of three pesticides (isoproturon, methomyl and oxamyl). The modes of pesticide-sorbent interactions were analyzed by means of two modeling calculations, the first one representing only the mixture of suberin monomers used in the sorption study, and the second one including glycerol to the mixture of suberin monomers, as a building block of the suberin molecule. The results indicated that the highest sorption capacity exhibited by the sorbent was for isoproturon (33%) being methomyl and oxamyl sorbed by the main suberin components to a lesser extent (3% and<1%, respectively). In addition to van der Waals interactions with the apolar region of sorbent and isoproturon, modeling calculations evidenced the formation of a hydrogen bond between the isoproturon NH group and a carboxylic oxygen atom of a suberin monomer. In the case of methomyl and oxamyl only weak van der Waals interactions stabilize the pesticide-sorbent adducts. The presence of glycerol in the model provoked significant changes in the interactions with isoproturon and methomyl.

New insights into the interactions between cork chemical components and pesticides. The contribution of π-π interactions, hydrogen bonding and hydrophobic effect.

The role of chemical components of cork in the sorption of several pesticides has been investigated. For this purpose raw cork and three cork extracted fractions (i.e. cork free of aliphatic extractives, cork free of all extractives and cork free of all extractives and suberin) were used as sorbent of three ionic pesticides (propazine, 2,4-dichlorophenoxy acetic acid (2,4-D) and alachlor) and five non-ionic pesticides (chlorpyrifos, isoproturon, metamitron, methomyl and oxamyl) with a logKow within the range -0.47 to 4.92. The effect of cations on the ionic pesticides, propazine and 2,4-D sorption was also analyzed. Results indicated that the highest yields were obtained for chlorpyrifos and alachlor sorption onto raw cork (>55%). After removal of aliphatic extractives sorption of all pesticides increased that ranged from 3% for propazine to 31% for alachlor. In contrast, removal of phenolic extractives caused a sorption decrease. Low sorption yields were obtained for hydrophobic pesticides such as metamitron, oxamyl and methomyl (<11%) by using all cork fractions and extremely low when using raw cork (<1%). FTIR analysis was useful to indicate that lignin moieties were the main components involved on the sorption process. Modelling calculations evidenced that π-stacking interactions with the aromatic groups of lignin play a major role in determining the adsorption properties of cork toward aromatic pesticides. Results presented in this paper gain insights into the cork affinities for pesticides and the interactions involved in the sorption process and also enables to envisage sorption affinity of cork for other organic pollutants.

An integrated approach to understanding the sorption mechanism of phenanthrene by cork.

Previous studies have shown the high sorption affinity of polycyclic aromatic hydrocarbons by cork. The aim of the present work is to go further by investigating the sorption mechanism of polycyclic aromatic hydrocarbons (exemplified by phenanthrene) on cork and the availability of the chemical components (i.e. lignin, suberin, holocellulose and extractives) to retain phenanthrene. Two approaches were integrated to reach this objective: (1) statistical multivariate analysis to obtain correlations between the sorption capacity, measured as K(oc), and the sorbent properties (i.e. polarity, acidic functional groups, %dichloromethane extractives, %ethanol and water extractives, %suberin, %lignin and %holocellulose) and (2) modeling calculations to obtain information on interaction at the molecular level. The statistical multivariate analysis demonstrated a strong and positive correlation between K(oc) and the lignin content as well as negative correlations between K(oc) and the phenolic groups and %dichloromethane extractives contents. The modeling study showed that the lignin-phenanthrene interaction is mostly hydrophobic in nature being largely determined by the π-stacking interaction between the aromatic groups of the interacting partners. This result justifies the observed correlations as dichloromethane extractives, being hydrophobic, compete with phenanthrene adsorption, whereas phenolic groups, as well as negatively charged groups, enhance the hydrophilic character of the sorbent surface, thus hindering the adsorption of phenanthrene.

Concentrations of organochlorine pesticides and 2,4,6-trichloroanisole in cork bark.

Organochlorine pesticides are persistent lipophilic organic pollutants and tend to accumulate in growing plants. During growth, cork is in contact with the open air for long periods (9-12 years). Owing to the previous widespread use of organochlorine pesticides and their high persistence in the environment, there is a risk that residues of such pesticides may be present in cork. In this study, the concentrations of 14 organochlorine pesticides-all of which are indicators of environmental pollution-were analyzed in cork bark samples from three regions in Spain and one in Portugal. In addition, the concentrations of 2,4,6-trichlorophenol (TCP) and 2,4,6-trichloroanisole (TCA) were also analyzed. Our results show only very low concentrations of lindane, γ-HCH (<2.6 ng g(-1)) and its byproducts α-HCH (<3.5 ng g(-1)) and ß-HCH (<0.6 ng g(-1)). Among the DDT and its metabolites, only two were found: p,p'-DDT was found in a cork sample from Extremadura (0.1 ng g(-1)) and p,p'-DDE was present at a maximum concentration of 2.9 ng g(-1) in a cork sample from Castile-La Mancha. However, all concentrations were well below the legal limit established by Regulation (EC) No. 396/2005 (10 ng g(-1) in foodstuffs). We can conclude, therefore, that the cork samples we studied complied with food safety standards.

Salicylic acid permeation: a comparative study with different vehicles and membranes

Considering the skin's function, different dermal pharmaceutical forms can be developed according to the type of therapeutic activity, active principle and excipients involved in the formulation, such as [quot ]transdermal preparations[quot ]. In the present study, the permeation parameters of the non-steroidal anti-inflammatory drug, salicylic acid (SA) through synthetic membrane, polyvinyliden difluoride, and a biological membrane, egg shell membrane, with different vehicles, propylene glycol, isopropyl alcohol and carbopol gel, were determined. The reported physicochemical parameters of SA from CG were significantly higher than those obtained using PG and IP. This is attributed to the lipophilic nature of the vehicle that facilitates the release and penetration of the active principle, thus acting sinergicall y. The permeation profiles of SA allow us to state that permeation kinetics is of first order, so that the flux values obtained are in direct proportion to the specific rates of drug release.

Salicylic acid permeation: a comparative study with different vehicles and membranes

Considering the skins function, different dermal pharmaceutical forms can be developed according to the type of therapeutic activity, active principle and excipients involved in the formulation, such as [quot ]transdermal preparations[quot ]. In the present study, the permeation parameters of the non-steroidal anti-inflammatory drug, salicylic acid (SA) through synthetic membrane, polyvinyliden difluoride, and a biological membrane, egg shell membrane, with different vehicles, propylene glycol, isopropyl alcohol and carbopol gel, were determined. The reported physicochemical parameters of SA from CG were significantly higher than those obtained using PG and IP. This is attributed to the lipophilic nature of the vehicle that facilitates the release and penetration of the active principle, thus acting sinergicall y. The permeation profiles of SA allow us to state that permeation kinetics is of first order, so that the flux values obtained are in direct proportion to the specific rates of drug release.(AU)

[Diagnosis and treatment of hepatic encephalopathy in Spain: results of a survey of hepatologists]. / Diagnóstico y tratamiento de la encefalopatía hepática en España: resultados de una encuesta dirigida a hepatólogos.

INTRODUCTION: Hepatic encephalopathy is a common complication of cirrhosis. Recent studies have challenged the efficacy of nonabsorbable disaccharides and have reported that protein restriction may pose risks to patients with cirrhosis and hepatic encephalopathy. AIM: To determine the diagnostic and therapeutic practices of physicians treating patients with hepatic encephalopathy. MATERIAL AND METHODS: We designed a 20-item questionnaire, which was mailed to the members of the Spanish Society for the Study of the Liver. RESULTS: We received 128 questionnaires, completed by physicians with wide clinical experience. They reported that the most common precipitating factors in episodic encephalopathy were infections (22%), diuretics (21%), and gastrointestinal bleeding (21%). The usual treatment of episodic encephalopathy was administration of nonabsorbable disaccharides (90%) and protein restriction (52%). Patients with chronic encephalopathy were also usually treated with nonabsorbable disaccharides (94%) and protein restriction (40%). Fifty-nine percent of the hepatologists never carried out neurophysiologic or neuropsychologic assessment for the diagnosis of minimal hepatic encephalopathy. CONCLUSION: Although their efficacy has been questioned, nonabsorbable disaccharides and protein restriction are the most commonly prescribed treatments for hepatic encephalopathy. Future studies are needed to assess the efficacy and risks of these treatments. Most hepatologists never assess minimal hepatic encephalopathy in patients with cirrhosis.

Diagnóstico y tratamiento de la encefalopatía hepática en España: resultados de una encuesta dirigida a hepatólogos / Diagnosis and treatment of hepatic encephalopathy in Spain: results of a survey of hepatologists

Introducción: La encefalopatía hepática es una complicación frecuente de la cirrosis hepática. Estudios recientes han cuestionado la eficacia de los disacáridos no absorbibles y han señalado que la restricción proteica podría tener riesgos en pacientes con cirrosis y encefalopatía hepática. Objetivo: Conocer los hábitos diagnósticos y terapéuticos de los médicos que tratan a pacientes con encefalopatía hepática. Material y métodos: Se elaboró un cuestionario de 20 preguntas que se envió por correo a los miembros de la Asociación Española para el Estudio del Hígado (AEEH). Resultados: Se recibieron 128 encuestas, completadas por médicos con larga experiencia. En referencia a la encefalopatía episódica, los encuestados consideraron que los factores precipitantes más frecuentes fueron las infecciones (22%), los diuréticos (21%) y la hemorragia digestiva (21%). El tratamiento habitual de la encefalopatía episódica consiste en la administración de disacáridos no absorbibles (90%) y en la restricción proteica (52%). La encefalopatía crónica también se trata habitualmente con disacáridos no absorbibles (94%), junto con restricción proteica (40%). Un 59% de los hepatólogos nunca realizan exploraciones neurofisiológicas o neuropsicológicas para el diagnóstico de la encefalopatía hepática mínima. Conclusión: A pesar de las dudas acerca de la eficacia de los disacáridos no absorbibles y de la restricción proteica, éstas son las medidas terapéuticas habituales en la encefalopatía hepática, lo que muestra la importancia de aclarar la eficacia y los riesgos de los disacáridos no absorbibles y de la restricción proteica. La mayoría de los hepatólogos no investigan nunca la encefalopatía hepática mínima en los pacientes con cirrosis hepática

Introduction: Hepatic encephalopathy is a common complication of cirrhosis. Recent studies have challenged the efficacy of nonabsorbable disaccharides and have reported that protein restriction may pose risks to patients with cirrhosis and hepatic encephalopathy. Aim: To determine the diagnostic and therapeutic practices of physicians treating patients with hepatic encephalopathy. Material and methods: We designed a 20-item questionnaire, which was mailed to the members of the Spanish Society for the Study of the Liver. Results: We received 128 questionnaires, completed by physicians with wide clinical experience. They reported that the most common precipitating factors in episodic encephalopathy were infections (22%), diuretics (21%), and gastrointestinal bleeding (21%). The usual treatment of episodic encephalopathy was administration of nonabsorbable disaccharides (90%) and protein restriction (52%). Patients with chronic encephalopathy were also usually treated with nonabsorbable disaccharides (94%) and protein restriction (40%). Fifty-nine percent of the hepatologists never carried out neurophysiologic or neuropsychologic assessment for the diagnosis of minimal hepatic encephalopathy. Conclusion: Although their efficacy has been questioned, nonabsorbable disaccharides and protein restriction are the most commonly prescribed treatments for hepatic encephalopathy. Future studies are needed to assess the efficacy and risks of these treatments. Most hepatologists never assess minimal hepatic encephalopathy in patients with cirrhosis

Diagnóstico y tratamiento de la encefalopatía hepática en España: resultados de una encuesta dirigida a hepatólogos / Diagnosis and treatment of hepatic encephalopathy in Spain: results of a survey of hepatologists

Introducción: La encefalopatía hepática es una complicación frecuente de la cirrosis hepática. Estudios recientes han cuestionado la eficacia de los disacáridos no absorbibles y han señalado que la restricción proteica podría tener riesgos en pacientes con cirrosis y encefalopatía hepática. Objetivo: Conocer los hábitos diagnósticos y terapéuticos de los médicos que tratan a pacientes con encefalopatía hepática. Material y métodos: Se elaboró un cuestionario de 20 preguntas que se envió por correo a los miembros de la Asociación Española para el Estudio del Hígado (AEEH). Resultados: Se recibieron 128 encuestas, completadas por médicos con larga experiencia. En referencia a la encefalopatía episódica, los encuestados consideraron que los factores precipitantes más frecuentes fueron las infecciones (22%), los diuréticos (21%) y la hemorragia digestiva (21%). El tratamiento habitual de la encefalopatía episódica consiste en la administración de disacáridos no absorbibles (90%) y en la restricción proteica (52%). La encefalopatía crónica también se trata habitualmente con disacáridos no absorbibles (94%), junto con restricción proteica (40%). Un 59% de los hepatólogos nunca realizan exploraciones neurofisiológicas o neuropsicológicas para el diagnóstico de la encefalopatía hepática mínima. Conclusión: A pesar de las dudas acerca de la eficacia de los disacáridos no absorbibles y de la restricción proteica, éstas son las medidas terapéuticas habituales en la encefalopatía hepática, lo que muestra la importancia de aclarar la eficacia y los riesgos de los disacáridos no absorbibles y de la restricción proteica. La mayoría de los hepatólogos no investigan nunca la encefalopatía hepática mínima en los pacientes con cirrosis hepática

Introduction: Hepatic encephalopathy is a common complication of cirrhosis. Recent studies have challenged the efficacy of nonabsorbable disaccharides and have reported that protein restriction may pose risks to patients with cirrhosis and hepatic encephalopathy. Aim: To determine the diagnostic and therapeutic practices of physicians treating patients with hepatic encephalopathy. Material and methods: We designed a 20-item questionnaire, which was mailed to the members of the Spanish Society for the Study of the Liver. Results: We received 128 questionnaires, completed by physicians with wide clinical experience. They reported that the most common precipitating factors in episodic encephalopathy were infections (22%), diuretics (21%), and gastrointestinal bleeding (21%). The usual treatment of episodic encephalopathy was administration of nonabsorbable disaccharides (90%) and protein restriction (52%). Patients with chronic encephalopathy were also usually treated with nonabsorbable disaccharides (94%) and protein restriction (40%). Fifty-nine percent of the hepatologists never carried out neurophysiologic or neuropsychologic assessment for the diagnosis of minimal hepatic encephalopathy. Conclusion: Although their efficacy has been questioned, nonabsorbable disaccharides and protein restriction are the most commonly prescribed treatments for hepatic encephalopathy. Future studies are needed to assess the efficacy and risks of these treatments. Most hepatologists never assess minimal hepatic encephalopathy in patients with cirrhosis

Polycyclic aromatic hydrocarbons in rainwater and surface waters of Lake Maggiore, a subalpine lake in Northern Italy.

Fourteen polycyclic aromatic hydrocarbons (PAHs) were measured in surface waters and precipitation inputs to Lake Maggiore, a subalpine lake in Northern Italy, from July 2003 to January 2004. Particulate and dissolved phases in surface water and rain samples were determined. Analyses of PAHs were performed using XAD-2 resin to isolate the dissolved PAHs and subsequent extraction by accelerated solvent extraction (ASE). Both the dissolved and particulate phase PAH patterns in surface water and rainwater samples were dominated by the low molecular weight compounds (e.g., phenanthrene, fluoranthene and pyrene). More than 85% of PAHs in surface waters and 72% of PAHs in rainwater were associated to the dissolved phase. The SigmaPAH concentrations in surface waters (particulate and dissolved phases) were 0.584 +/- 0.033 ng l(-1), 2.9 +/- 0.312 ng l(-1) and in rainwater (particulate and dissolved phases) 27.5 +/- 2 ng l(-1), 75.4 +/- 9 ng l(-1), respectively. Temporal variability of PAH concentrations in rain and surface water samples were observed, with higher concentrations in November and December, coinciding with the largest precipitation amounts. The comparison of PAH signatures in rainwater and surface waters seems to indicate that wet deposition (2.5-41 microg m(-2) month(-1)) is the main source of PAH contamination into surface waters of Lake Maggiore.

Distribution of polycyclic aromatic hydrocarbons in riverine waters after Mediterranean forest fires.

Extensive forest fires occurred in Catalonia, northern Spain, in 1994. In our study, concentrations and profiles of 12 parent polycyclic aromatic hydrocarbons (PAHs) were determined in riverine waters, ash and sediment samples at nine sampling sites (W1-W9) and at three sampling dates from Llobregat hydrographic basin: in August, 1994, one month after the extensive forest fires; in September, 1994, after the first heavy autumn rainfalls and in January, 1995, six months after forest fires. In August 1994, the total concentrations of 12 PAHs measured in riverine waters varied from 2 ng/l to 336 ng/l. In September 1994, the total PAH concentrations decreased to 0.2-31 ng/l and in January 1995, from 9 ng/l to 73 ng/l. In August, the composition pattern of PAHs showed a distribution dominated by 4-ring PAHs (pyrene, chrysene+triphenylene, benzo(a)anthracene) at W3-W6, W8 and W9 and 3-ring PAHs (phenanthrene) at W1, W2 and W7. In September, a preference by 3-ring PAHs (phenanthrene) at all sampling sites except W5 was shown and in January was clearly dominated by 4-ring PAHs (chrysene+triphenylene, pyrene, benzo(a)anthracene) at all sampling sites. In ash and sediment samples, the total concentrations of 12 PAHs ranged from 1.3 ng/g to 19 ng/g. The dominant compound was phenanthrene.

Isolation and analysis of polycyclic aromatic hydrocarbons from natural water using accelerated solvent extraction followed by gas chromatography-mass spectrometry.

An innovative analytical procedure for the analysis of polycyclic aromatic hydrocarbons (PAHs) from large-volume water samples is presented. It involves sample preparation, sampling and the elution process in an automated continuous procedure involving the ASE technique. Prior to sampling, a XAD-2 resin column is prepared on the basis of a commercial accelerated solvent extraction (ASE) cartridge so that the resin bed is permanently fixed. Then, the XAD column inside the ASE cartridge is cleaned and conditioned. The sampling procedure involves conventional filtration with subsequent isolation of dissolved PAHs on an XAD-2 resin contained in the ASE cartridge. After sampling, the XAD-2 resin content inside the cartridge is eluted by ASE without any further sample preparation and subsequently reused. In order to validate the procedure, the PAHs were isolated from water samples from the Lake Maggiore (North of Italy) using both XAD-2 resin adsorption and hexane liquid-liquid extraction according to the International Standard Methodology ISO 17993. The mean percentages of deviation between concentrations obtained by both methodologies range from 6% for benzo(a)pyrene to 15% for fluoranthene and benzo(b,k)fluoranthene. Compared to the traditional techniques, this procedure offers numerous practical advantages: easy to perform, fast, savings in solvent volume and in time, all steps are fully automated thus avoiding any XAD-2 resin manipulation during and between steps and moreover, low detection limits were provided (0.001 ng l(-1) for chrysene, benzo(b,k)fluoranthene, benzo(a)pyrene, dibenz(a,h)anthracene, benzo(g,h,i)perylene and indeno(1,2,3-cd)pyrene, and 0.01 ng l(-1) for acenaphthylene and fluoranthene). This procedure was developed in the frame of a project aimed at evaluating the diffuse input of organic contaminants in the Lake Maggiore.

Trace analysis of polycyclic aromatic hydrocarbons in suspended particulate matter by accelerated solvent extraction followed by gas chromatography-mass spectrometry.

An analytical procedure based on extraction by accelerated solvent extraction (ASE) followed by gas chromatography-mass spectrometry (GC/MS) analysis has been developed for the determination of particulate polycyclic aromatic hydrocarbons (PAHs) from large-volume water samples (20 L). The effect of temperature and number of cycles on the efficiency of ASE was investigated: the best results were obtained by using a temperature of 100 degrees C and one static cycle. A mixture of hexane/acetone 1:1 (v/v) was used as extraction solvent. Mean total method recovery under optimized conditions was 85%. The developed methodology was applied to the analysis of suspended particulate matter from Lake Maggiore waters (north of Italy). Mean PAH concentrations in suspended particulate matter from Lake Maggiore ranged from 0.2 ng L(-1) for anthracene to 18.7 ng L(-1) for naphthalene.

Estudio multicéntrico sobre estigma, necesidades y cuidados de personas con trastornos mentales de larga evolución / Multi-centered study on the stigma, needs, and care of persons with long-evolving mental disorder

Introducción: El estudio multicéntrico de la red MARISTAN tiene como objetivo estudiar el estigma, las necesidades y los cuidados no formales relacionados con las personas que padecen de trastornos psicóticos de larga evolución. Método: El estudio, en su fase inicial, utiliza la técnica de grupos focales con el objeto de obtener una percepción de los tres aspectos señalados desde tres perspectivas complementarias: de los usuarios, de los familiares que les atienden y de los profesionales que les brindan los cuidados formales. Discusión: Aún se encuentra en fase de análisis de resultados, sin embargo revela su importancia desde el punto de vista transcultural y de la sistematización de las diferentes fases de la investigación cualitativa (AU)

Computational model of the complex between GR113808 and the 5-HT4 receptor guided by site-directed mutagenesis and the crystal structure of rhodopsin.

A computational model of the transmembrane domain of the human 5-HT4 receptorcomplexed with the GR113808 antagonist was constructed from the crystal structure of rhodopsin and the putative residues of the ligand-binding site, experimentally determined by site-directed mutagenesis. The recognition mode of GR113808 consist of: (i) the ionic interaction between the protonated amine and Asp3.32; (ii) the hydrogen bond between the carbonylic oxygen and Ser5.43; (iii) the hydrogen bond between the ether oxygen and Asn6.55; (iv) the hydrogen bond between the C-H groups adjacent to the protonated piperidine nitrogen and the pi electrons of Phe6.51; and (v) the pi-sigma aromatic-aromatic interaction between the indole ring and Phe6.52. This computational model offers structural indications about the role of Asp3.32, Ser5.43, Phe6.51, Phe6.52, and Asn6.55 in the experimental binding affinities. Asp3.32Asn mutation does not affect the binding of GR113808 because the loss of binding affinity from an ion pair to a charged hydrogen bond is compensated by the larger energetical penalty of Asp to disrupt its side chain environment in the ligand-free form, and the larger interaction between Phe6.51 and the piperidine ring of the ligand in the mutant receptor. In the Phe6.52Val mutant the indole ring of the ligand replaces the interaction with Phe6.52 by a similarly intense interaction with Tyr5.38, with no significant effect in the binding of GR113808. The mutation of Asn6.55 to Leu replaces the hydrogen bond of the ether oxygen of the ligand from Asn6.55 to Cys5.42, with a decrease of binding affinity that approximately equals the free energy difference between the SH...O and NH...O hydrogen bonds. Because these residues are also present in the other members of the neurotransmitter family of G protein-coupled receptors, these findings will also serve for our understanding of the binding of related ligands to their cognate receptors.

Serine and threonine residues bend alpha-helices in the chi(1) = g(-) conformation.

The relationship between the Ser, Thr, and Cys side-chain conformation (chi(1) = g(-), t, g(+)) and the main-chain conformation (phi and psi angles) has been studied in a selection of protein structures that contain alpha-helices. The statistical results show that the g(-) conformation of both Ser and Thr residues decreases their phi angles and increases their psi angles relative to Ala, used as a control. The additional hydrogen bond formed between the O(gamma) atom of Ser and Thr and the i-3 or i-4 peptide carbonyl oxygen induces or stabilizes a bending angle in the helix 3-4 degrees larger than for Ala. This is of particular significance for membrane proteins. Incorporation of this small bending angle in the transmembrane alpha-helix at one side of the cell membrane results in a significant displacement of the residues located at the other side of the membrane. We hypothesize that local alterations of the rotamer configurations of these Ser and Thr residues may result in significant conformational changes across transmembrane helices, and thus participate in the molecular mechanisms underlying transmembrane signaling. This finding has provided the structural basis to understand the experimentally observed influence of Ser residues on the conformational equilibrium between inactive and active states of the receptor, in the neurotransmitter subfamily of G protein-coupled receptors.

Colorimetry of diaphorase in commercial preparations and clinical chemical reagents by use of tetrazolium salts.

We describe a procedure for assay of diaphorase activity in commercial purified preparations and in clinical chemical reagents by use of iodonitrotetrazolium chloride or other tetrazolium salts. The method is based on measurement of the formazan produced by enzymic reduction of tetrazolium salts in the presence of NADH. The assay procedure has been optimized for linear kinetics, simplicity of operation, nondetectable blank rates, and extended activity/enzyme concentration proportionality. The proposed method has several advantages over the older assay by use of dichlorophenolindophenol.