Delphi consensus on the diagnosis and treatment of patients with short stature in Spain: GROW-SENS study.

PURPOSE: To identify consensus aspects related to the diagnosis, monitoring, and treatment of short stature in children to promote excellence in clinical practice. METHODS: Delphi consensus organised in three rounds completed by 36 paediatric endocrinologists. The questionnaire consisted of 26 topics grouped into: (1) diagnosis; (2) monitoring of the small-for-gestational-age (SGA) patient; (3) growth hormone treatment; and (4) treatment adherence. For each topic, different questions or statements were proposed. RESULTS: After three rounds, consensus was reached on 16 of the 26 topics. The main agreements were: (1) diagnosis tests considered as a priority in Primary Care were complete blood count, biochemistry, thyroid profile, and coeliac disease screening. The genetic test with the greatest diagnostic value was karyotyping. The main criterion for initiating a diagnostic study was prediction of adult stature 2 standard deviations below the target height; (2) the main criterion for initiating treatment in SGA patients was the previous growth pattern and mean parental stature; (3) the main criterion for response to treatment was a significant increase in growth velocity and the most important parameter to monitor adverse events was carbohydrate metabolism; (4) the main attitude towards non-responding patients is to check their treatment adherence with recording devices. The most important criterion for choosing the delivery device was its technical characteristics. CONCLUSIONS: This study shows the different degrees of consensus among paediatric endocrinologists in Spain concerning the diagnosis and treatment of short stature, which enables the identification of research areas to optimise the management of such patients.

Sorption, degradation and transport phenomena of alcohol ethoxysulfates in agricultural soils. Laboratory studies.

In the present work, laboratory studies were conducted in order to determine and model the sorption, degradation and transport processes of alcohol ethoxysulfates (AES), one of the most important groups of anionic surfactants. Adsorption/desorption isotherms were obtained for several structurally related AES ethoxymers (homologue AES-C12En with n = 0-10 ethoxymer units and homologue AES-C14En with n = 0-7 ethoxymer units) using a batch equilibrium method. Data were fitted to a linear and a Freundlich isotherm models. Additionally, experiments in continuous-flow soil columns were also carried out and the breakthrough curves observed for each compound were studied. Breakthrough curves were used to determine the fundamental parameters of the transport model (hydrodynamic dispersion coefficient, degradation rate constant and adsorption/desorption isotherm slope), that is the main phenomena that take place simultaneously when AES move through agricultural soil. When the results obtained for the AES ethoxymers are combined, they reveal a clear and consistent trend towards a sorption increase with the number of ethoxylated units and with the length of the alkyl chain that opens the possibility to estimate the values of the transport parameters for other structurally related ethoxymers.

Clinical and genetic characterization of congenital hyperinsulinism in Spain.

CONTEXT: Congenital hyperinsulinism (CHI) is a clinically and genetically heterogeneous disease characterized by severe hypoglycemia caused by inappropriate insulin secretion by pancreatic ß-cells. OBJECTIVE: To characterize clinically and genetically CHI patients in Spain. DESIGN AND METHODS: We included 50 patients with CHI from Spain. Clinical information was provided by the referring clinicians. Mutational analysis was carried out for KCNJ11, ABCC8, and GCK genes. The GLUD1, HNF4A, HNF1A, UCP2, and HADH genes were sequenced depending on the clinical phenotype. RESULTS: We identified the genetic etiology in 28 of the 50 CHI patients tested: 21 had a mutation in KATP channel genes (42%), three in GLUD1 (6%), and four in GCK (8%). Most mutations were found in ABCC8 (20/50). Half of these patients (10/20) were homozygous or compound heterozygous, with nine being unresponsive to diazoxide treatment. The other half had heterozygous mutations in ABCC8, six of them being unresponsive to diazoxide treatment and four being responsive to diazoxide treatment. We identified 22 different mutations in the KATP channel genes, of which ten were novel. Notably, patients with ABCC8 mutations were diagnosed earlier, with lower blood glucose levels and required higher doses of diazoxide than those without a genetic diagnosis. CONCLUSIONS: Genetic analysis revealed mutations in 56% of the CHI patients. ABCC8 mutations are the most frequent cause of CHI in Spain. We found ten novel mutations in the KATP channel genes. The genetic diagnosis is more likely to be achieved in patients with onset within the first week of life and in those who fail to respond to diazoxide treatment.

Sitosterolemia en paciente de 2 años con xantomas y alteraciones hematológicas / Sitosterolemia in a 2 year-old child with xanthomas and hematological anomalies

No disponible

Identification of prometon, deisopropylprometon, and hydroxyprometon in groundwater by high resolution liquid chromatography/mass spectrometry.

Prometon, a major soil sterilant, and its main transformation products, deisopropylprometon (N(2)-isopropyl-6-methoxy-1,3,5-triazine-2,4-diamine) and hydroxyprometon (4,6-bis(isopropylamino)-1,3,5-triazin-2-ol), were identified as the dominant triazine herbicides in groundwater samples from 51 locations in Colorado, USA, over a two-year time period. They were concentrated from water by solid phase extraction and detected using an ultrahigh pressure, liquid chromatography-quadrupole time of flight tandem mass spectrometry (UHPLC/QTOF-MS). The transformation products, deisopropylprometon and hydroxyprometon, were confirmed using MS-MS experiments. An original strategy was applied to form the degradation standards for deisopropylprometon and hydroxyprometon, which consisted of photo-degradation of prometon followed by MS-MS analysis. The concentration of prometon ranged from the detection limit of 3 ng·L(-1) to 87 ng·L(-1), hydroxyprometon ranged up to 50 ng·L(-1), and deisopropylprometon up to 100 ng·L(-1), with a frequency of detection of 80%, which was greater than the other triazines detected in the groundwater samples. A new ratio is proposed for prometon degradation called the "deisopropylprometon to prometon ratio" or the DIP ratio, as an indicator of prometon residence time in groundwater. Furthermore, these data suggest that prometon is more of an issue for groundwater contamination in urban areas rather than agricultural areas.

New method for the determination of carbamate and pyrethroid insecticides in water samples using on-line SPE fused core column chromatography.

A new HPLC column-switching method using large volume sample injection and fused-core columns for on-line solid phase extraction have been developed for the determination of the following carbamates and pyrethroids: aldicarb, carbaryl, pirimicarb, carbofuran, kadethrin, flumethrin, fenpropathrin, fenoxycarb, tau-fluvalinate and fenvalerate, in surface water samples. Sudan I was used as internal standard. The proposed method was performed using 100 µl sample injection followed by an on-line solid phase extraction procedure and finally the compounds were identified and quantified by liquid chromatography with ultraviolet detection. The separation was carried out on C-18 reversed phase column based on fused-core particle technology. The influence of the injected sample volume, the variables affecting to SPE process and the conditions for the separation on an analytical column, were studied and optimized. The limits of detection ranged from 5.5 to 8.9 µg L(-1), and limits of quantification from 18.4 to 29.7 µg L(-1), while inter- and intra-day variability was under 15%. This new analytical procedure was satisfactorily applied for the determination of these organic pollutants in surface water samples located in Czech Republic. Concentration levels were found for some of these pollutants up to 26.11 µg L(-1) in the river Elbe and up to 34.53 µg L(-1) in the closed lakes samples.

Evaluation of the levels of alcohol sulfates and ethoxysulfates in marine sediments near wastewater discharge points along the coast of Tenerife Island.

Alcohol sulfates (AS) and alcohol ethoxysulfates (AES) are all High Production Volume and 'down-the-drain' chemicals used globally in detergent and personal care products, resulting in low levels ultimately released to the environment via wastewater treatment plant effluents. They have a strong affinity for sorption to sediments. Almost 50% of Tenerife Island surface area is environmentally protected. Therefore, determination of concentration levels of AS/AES in marine sediments near wastewater discharge points along the coast of the Island is of interest. These data were obtained after pressurized liquid extraction and liquid chromatography-tandem mass spectrometry analysis. Short chains of AES and especially of AS dominated the homologue distribution for AES. The Principal Components Analysis was used. The results showed that the sources of AS and AES were the same and that both compounds exhibit similar behavior. Three different patterns in the distribution for homologues and ethoxymers were found.

Determination of alcohol sulfates and alcohol ethoxysulfates in marine and river sediments using liquid chromatography-tandem mass spectrometry.

A novel and successful method has been developed for the identification and quantification of alcohol sulfates (AS) homologues and alcohol ethoxysulfates (AES) ethoxymers in marine and river sediment samples. The method involves the extraction of 5.00 g of dry sample with methanol using pressurized liquid extraction (PLE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). 2-Octylbenzene sulfonic acid sodium salt (2ØC8-LAS) was used as internal standard. The analytical methods were applied to marine sediments collected from the coast of Almeria (South-east Spain) and river sediments collected from the Monachil river (Granada, South-east Spain). For AS homologues, the found limits of detection were 0.04-0.08 µg g(-1) for marine and river sediments. For AES ethoxymers, the found limits of detection were 0.03-0.09 µg g(-1) and 0.06-0.22 µg g(-1) for marine and river sediments, respectively. The highest concentrations of AS and AES were found in river sediment samples. Significant differences were also observed between the behavior of short-chain compounds (C12) and long-chain compounds (C14 to C18). The influence of the physic-chemical properties of water on the occurrence of these compounds was also evaluated, and differences between long- and short-chain compounds were also observed. Additionally, principal components analyses were carried out in order to study the relationship between variables and to evaluate the sources of data variability and behavior patterns. Finally, important conclusions were drawn regarding the environmental behavior of AS and AES.

A new procedure of determination of alcohol sulfates and alcohol ethoxysulfates in agricultural soils.

The number of analytical methodologies that focus in the determination of alcohol sulfates (AS) and alcohol ethoxysulfates (AES) in terrestrial environment is very limited. In the present work, a new methodology to improve the extraction and determination of AS and AES in agricultural soil samples has been developed. Prior to instrumental analysis, an extraction procedure using pressurized liquid extraction with methanol (PLE) was carried out in order to obtain the highest recoveries and improve sensitivity. The most influential variables affecting the PLE procedure were optimized. Then, the separation and quantification of analytes were performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The limits of detection (LOD) ranged from 0.03 to 0.08µgg(-1) for AS homologues and in the case of AES ethoxymers from 0.03 to 0.09µgg(-1) for AES-C12Ex and from 0.03 to 0.08µgg(-1) for AES-C14Ex. Matrix-matched calibration was used. Trueness was evaluated by using a spike recovery assay with spiked blank samples, and the recoveries ranged from 98.3% to 101.0% for AS and from 99.9% to 100.1% for AES. The method was satisfactorily applied in a field study designed to evaluate the environmental behavior of these compounds in agricultural soil.

Determination of alcohol sulfates in wastewater treatment plant influents and effluents by gas chromatography-mass spectrometry.

In the present paper, we developed an accurate method for the analysis of alcohol sulfates (AS) in wastewater samples from wastewater treatment plant (WWTP) influents and effluents. Although many methodologies have been published in the literature concerning the study of anionic surfactants in environmental samples, at present, the number of analytical methodologies that focus in the determination of AS by gas chromatography in the different environmental compartments is limited. The reason for this is that gas chromatography-mass spectrometry (GC-MS) technique requires a previous hydrolysis reaction followed by derivatization reactions. In the present work, we proposed a new procedure in which the hydrolysis and derivatization reactions take place in one single step and AS are directly converted to trimethylsilyl derivatives. The main factors affecting solid-phase extraction (SPE), hydrolysis/derivatization and GC-MS procedures were accurately optimised. Quantification of the target compounds was performed by using GC-MS in selected ion monitoring (SIM) mode. The limits of detection (LOD) obtained ranged from 0.2 to 0.3 µg L(-1), and limits of quantification (LOQ) from 0.5 to 1.0 µg L(-1), while inter- and intra-day variability was under 5%. A recovery assay was also carried out. Recovery rates for homologues in spiked samples ranged from 96 to 103%. The proposed method was successfully applied for the determination of anionic surfactants in wastewater samples from one WWTP located in Granada (Spain). Concentration levels for the homologues up to 39.4 µg L(-1) in influent and up to 8.1 µg L(-1) in effluent wastewater samples.

[Spanish cross-sectional growth study 2008. Part II. Height, weight and body mass index values from birth to adulthood]. / Estudio transversal español de crecimiento 2008. Parte II: valores de talla, peso e índice de masa corporal desde el nacimiento a la talla adulta.

INTRODUCTION: In developed countries a secular trend in growth has been reported. Our aim was to evaluate weight, height and body mass index (BMI) values in a Spanish population coming from Andalusia, Barcelona, Bilbao and Zaragoza, and to compare these values with those obtained before 1988 (BIB 88 and CAT 87 studies). SUBJECTS AND METHODS: Cross-sectional evaluation of height, weight and BMI in 32,064 subjects (16,607 males and 15,457 females) from birth to adulthood between the years 2000 and 2004. Three subpopulations were evaluated: a) 5,796 (2,974 males, 2,822 females) newborns at term from normal gestations; b) 23,701 (12,358 males; 11,343 females) children and adolescents 0.25-18 years old, and c) 2,567 (1,275 males, 1,292 females) young adults 18.1-24 years of age. All were healthy caucasians, and their parents from Spanish origin. The LSM method was used. RESULTS: Mean, standard deviation, Skewness index and percentiles values with a 0.25-0.5 year-period intervals from birth to adulthood are reported. As regards the data obtained previously in Spanish populations, an increase of 1.8 cm, 1.4 cm and 3.3 cm were observed in adult height for percentiles 3, 50 and 97 in males respect to BIB 88 and 2.5 cm, 3.3 cm and 3.8 respect to CAT 87. In females these values were 3.5 cm, 2.5 cm and 4.2 cm respect to BIB 88 and 3.5 cm, 3.1 cm and 3.9 cm respect to CAT 87. The corresponding values for weight, in males, were increased in 5.4 kg, 6.2 kg and 11.7 kg respect to BIB 88 and 6.7 kg, 6.3 kg and 10.1 kg respect to CAT 87; in females these increased were 1.7 kg, 2,2 kg and 8.3 kg respect to BIB 88 and 1.8 kg, 2.4 kg and 3.6 kg respect to CAT 87. The corresponding increased for BMI values, in males, were 2.0, 1.4 and 3.9 respect to BIB 88 and 0.1, 0.2 and 5.3 respect to CAT 87; in females these values were 0.9, 0.4 and 3.7 respect to BIB 88 and 1.8, 0.1 and 4 respect to CAT 87. In young adults, 25 and 30 BMI values correspond to percentiles 80 and 97 in males, and 85 and 97 in females. Mean values of adult height were similar to those observed in other longitudinal and cross-sectional Spanish, European, and American studies, but lower than those reported for German, Swedish and Netherlands populations. CONCLUSIONS: A secular trend of growth was observed in our population with a non-proportional increased of weight to height ratio (BMI) values, particularly for those corresponding to the 97 percentile. The need of periodical updates of growth data used in the evaluation of children and adolescents is required.

Estudio transversal español de crecimiento 2008. Parte II: valores de talla, peso e índice de masa corporal desde el nacimiento a la talla adulta / Spanish cross-sectional growth study 2008. Part II. Height, weight and body mass index values from birth to adulthood

Introducción: En las sociedades desarrolladas existe una aceleración secular del crecimiento. Entre los años 2000 y 2004 hemos valorado el crecimiento en una población caucásica española procedente de Andalucía, Barcelona, Bilbao y Zaragoza y hemos comparado los resultados con estudios españoles realizados antes de 1988 en poblaciones caucásicas de Bilbao (BIB 88) y Cataluña (CAT 87). Sujetos y métodos: Estudio transversal que valora el peso, la longitud y el índice de masa corporal (IMC) en 32.064 sujetos (16.607 varones y 15.457 mujeres) desde el nacimiento a la talla adulta: a) 5.796 son recién nacidos a término (2.974 varones y 2.822 mujeres) hijos de madres sanas, de gestaciones únicas; b) 23.701 son niños y adolescentes (12.358 varones y 11.343 mujeres) de 0,25 a 18 años de edad ambos inclusive, y c) 2.567 son adultos jóvenes (1.275 varones y 1.292 mujeres) de 18,1 a 24 años de edad. Todos estaban sanos, eran de raza caucásica y sus padres, de origen español. La distribución percentilada, el valor z-score y el diseño de las gráficas se ha realizado según el método LMS. Resultados: Se expresan los valores de la media, desviación estándar, coeficiente de Skewness y percentiles desde el nacimiento a la edad adulta, en intervalos de 0,25-0,50 años. Existe un dimorfismo sexual y un incremento en los valores de peso y talla de todos los percentiles respecto a los estudios BIB 88 y CAT 87. Los valores de los percentiles 3, 50 y 97 de la talla adulta son superiores en 1,8, 1,4 y 3,3 cm respecto a BIB 88, y en 2,5, 3,3 y 3,8 cm respecto a CAT 87 en los varones, y 3,5, 2,5 y 4,2 cm respecto a BIB 88 y 3,5, 3,1 y 3,9 cm respecto a CAT 87 en las mujeres. Los correspondientes valores de peso son 5,4, 6,2 y 11,7 kg superiores a los de BIB 88 y 6,7, 6,3 y 10,1 kg superiores a los de CAT 87 en los varones y 1,7, 2,2 y 8,3 kg superiores a los de BIB 88, y 1,8, 2,4 y 3,6 kg superiores CAT 87 en las mujeres. Los respectivos incrementos en el IMC son 2,0, 1,4 y 3,9 respecto a BIB 88 y –0,1, –0,2 y 5,3 respecto a CAT 87 en los varones y 0,9, 0,4 y 3,7 respecto a BIB 88 y –1,8, –0,1 y 4,0 respecto a CAT 87, en las mujeres. Los valores del IMC de 25 y 30 “en la edad adulta” corresponden a los percentiles 80 y 97 en los varones y 85 y 97 en las mujeres. En ambos sexos los valores de la talla media adulta son similares a los observados en otros estudios longitudinales y transversales españoles recientes y a los observados en estudios europeos y americanos, aunque inferiores a los de la población alemana, sueca y holandesa. Conclusiones: Con relación a estudios españoles previos, existe una aceleración secular de peso y talla, con un incremento desproporcionado en los valores del IMC correspondientes al percentil 75 o superiores, y de forma marcada en los del percentil 97. Este estudio muestra la necesidad de actualizar periódicamente los datos de referencia utilizados en la valoración del crecimiento durante la infancia y adolescencia (AU)

Introduction: In developed countries a secular trend in growth has been reported. Our aim was to evaluate weight, height and body mass index (BMI) values in a Spanish population coming from Andalusia, Barcelona, Bilbao and Zaragoza, and to compare these values with those obtained before 1988 (BIB 88 and CAT 87 studies). Subjects and methods: Cross-sectional evaluation of height, weight and BMI in 32,064 subjects (16,607 males and 15,457 females) from birth to adulthood between the years 2000 and 2004. Three subpopulations were evaluated: a) 5,796 (2,974 males, 2,822 females) newborns at term from normal gestations; b) 23,701 (12,358 males; 11,343 females) children and adolescents 0.25-18 years old, and c) 2,567 (1,275 males, 1,292 females) young adults 18.1-24 years of age. All were healthy caucasians, and their parents from Spanish origin. The LSM method was used. Results: Mean, standard deviation, Skewness index and percentiles values with a 0.25-0.5 year-period intervals from birth to adulthood are reported. As regards the data obtained previously in Spanish populations, an increase of 1.8 cm, 1.4 cm and 3.3 cm were observed in adult height for percetiles 3, 50 and 97 in males respect to BIB 88 and 2.5 cm, 3.3 cm and 3.8 respect to CAT 87. In females these values were 3.5 cm, 2.5 cm and 4.2 cm respect to BIB 88 and 3.5 cm, 3.1 cm and 3.9 cm respect to CAT 87. The corresponding values for weight, in males, were increased in 5.4 kg, 6.2 kg and 11.7 kg respect to BIB 88 and 6.7 kg, 6.3 kg and 10.1 kg respect to CAT 87; in females these increased were 1.7 kg, 2,2 kg and 8.3 kg respect to BIB 88 and 1.8 kg, 2.4 kg and 3.6 kg respect to CAT 87. The corresponding increased for BMI values, in males, were 2.0, 1.4 and 3.9 respect to BIB 88 and –0.1, –0.2 and 5.3 respect to CAT 87; in females these values were 0.9, 0.4 and 3.7 respect to BIB 88 and –1.8, –0.1 and 4 respect to CAT 87. In young adults, 25 and 30 BMI values correspond to percentiles 80 and 97 in males, and 85 and 97 in females. Mean values of adult height were similar to those observed in other longitudinal and cross-sectional Spanish, European, and American studies, but lower than those reported for German, Swedish and Netherlands populations. Conclusions: A secular trend of growth was observed in our population with a non-proportional increased of weight to height ratio (BMI) values, particularly for those corresponding to the 97 percentile. The need of periodical updates of growth data used in the evaluation of children and adolescents is required (AU)

In vitro culture and biochemical characterization of six trypanosome isolates from Peru and Brazil.

Six trypanosomatids isolated from different geographical areas from South America (Peru and Brazil) and different vectors and reservoir hosts (the triatomine Panstrongylus chinai [TP1], Triatoma infestans [TP2], Rhodnius ecuadorensis [TP3], R. prolixus [TB1], Didelphys marsupialis [TB2]), and one from a human asymptomatic patient [TB3], were characterized using lectin agglutination, isoenzyme profile, in vitro culture final metabolite patterns, and compared with a reference strain (Trypanosoma cruzi, Maracay strain [TC]). The different isolates were cultured in vitro in Grace's medium supplemented with 10% inactivated bovine foetal serum. According to our results and the statistical study, the isolate obtained from R. ecuadorensis should be designed as a Trypanosoma rangeli sp., showing all other isolates strong similarities to T. cruzi. Between them, two clusters could be identified, strongly correlating with the geographical origin. Cluster I grouped isolates from Peru and T. cruzi reference strain, and cluster II grouped the three Brazilian isolates.

In vitro evaluation of newly synthesised [1,2,4]triazolo[1,5a]pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli.

The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.

Biochemical and ultrastructural alterations caused by newly synthesized 1,2,4-triazole[1,5a]pyrimidine derivatives against Phytomonas staheli (Trypanosomatidae).

Six compounds, all newly synthesized triazole-pyrimidine derivatives that proved inhibitory of in in vitro growth of epimastigotes in Trypanosoma cruzi and of promastigotes of Leishmania donovani and Phytomonas staheli, were studied to investigate their toxic effects. As a biological model, the plant trypanosome P. staheli, which causes sudden wilt in the oil palm and Hartrot in the coconut palm, was used. The six compounds markedly inhibited macromolecule synthesis (nucleic acids and proteins) by the parasite. The cells treated with these compounds present severe damage in their ultrastructure-intense 'vacuolization, and appearance of lysosomes as well as other residual bodies. The mitochondrial section appeared larger in size. with a swollen matrix. In addition, these compounds changed the excretion of end metabolites, primarily affecting ethanol and acetate excretion, possibly by directly influencing certain enzymes (alcohol dehydrogenase and acetate synthetase) or their synthesis. 2000 Elsevier Science Ltd.

Biochemical characterization of a trypanosomatid isolated from the plant Amaranthus retroflexus.

A protozoan flagelate has recently been isolated from Amaranthus retroflexus. This plant grows near economically important crops in southeastern Spain, which are known to be parasitized by Phytomonas spp. The present study focuses on the characterization of the energy metabolism of this new isolate. These flagellates utilize glucose efficiently as their primary energy source, although they are unable to completely degrade it. They excrete ethanol, acetate, glycine, and succinate in lower amount, as well as ammonium. The presence of glycosomes was indicated by the early enzymes of the glycolytic pathway, one enzyme of the glycerol pathway (glycerol kinase), and malate dehydrogenase. No evidence of a fully functional citric-acid cycle was found. In the absence of catalase activity, these flagellates showed significant superoxide dismutase activity located in the glycosomal and cytosolic fractions. These trypanosomes, despite being morphologically and metabolically similar to other Phytomonas isolated from the same area, showed significant differences, suggesting that they are phylogenetically different species.

Trypanosomatid protozoa in plants of southeastern Spain: characterization by analysis of isoenzymes, kinetoplast DNA, and metabolic behavior.

Three flagellates of the family Trypanosomatidae were isolated from mango fruits (Mangifera indica) and from the stems of clover (Trifolium glomeratum) and Amaranth (Amaranthus retroflexus) in southeastern Spain and were adapted to in vitro culture in monophase media. The parasites showed an ultrastructural pattern similar to that of other species of the genus Phytomonas. Mango and clover isolates differed from amaranth isolates in ultrastructural terms. The isolates were characterized by isoenzymatic analysis and by kDNA analysis using five different restriction endonucleases. With eight of the nine enzymatic systems, mango and clover isolates were distinguished from those of amaranth. Nevertheless, with the enzymes malate dehydrogenase and superoxide dismutase, flagellates isolated from clover were differentiated from those isolated from mango. Electrophoretic and restriction-endonuclease analysis of kDNA minicircles showed similar restriction cleavage patterns for the isolates from mango and clover, whereas the patterns of the amaranth isolates differed. The results of the present study confirm that the strains isolated from mango and clover constitute a phylogenetically closely related group of plant trypanosomatids, which is more distantly related to the strain isolated from amaranth. The similarities in the results obtained for isolates from mango and clover foliage, on the one hand, and those obtained from tomato and cherimoya fruits (studied previously), on the other, as well as the geographic proximity of the different plants support the contention that only one strain is involved, albeit one strain that can parasitize different plants. Furthermore, some of the plants appear to act as reservoirs for the parasites. On the other hand, the metabolism studies using [1H]-nuclear magnetic resonance spectroscopy did not reveal that the catabolism of Phytomonas in general follows a pattern common to all the species or isolates. Phytomonas are incapable of completely degrading glucose, excreting a large part of their carbon skeleton into the medium as fermentative metabolites (acetate, ethanol, glycine, glycerol, and succinate).