Phytotoxic and antimicrobial activity of volatile and semi-volatile organic compounds from the endophyte Hypoxylon anthochroum strain Blaci isolated from Bursera lancifolia (Burseraceae).

AIMS: To evaluate the phytotoxic, antifungal and antioomycete activity; and, determine the chemical composition of the volatile organic compounds (VOCs) and semi-volatile metabolites produced by the endophyte Hypoxylon anthochroum strain Blaci isolated from Bursera lancifolia. METHODS AND RESULTS: Based on its macro- and micro-morphological features, the strain Blaci was identified as Nodulisporium sp.; partial analysis of its ITS1-5.8-ITS2 ribosomal gene sequence revealed the identity of the teleomorphic stage of the fungus as H. anthochroum. Phytotoxic and antimicrobial activities of VOCs, and culture medium and mycelium organic extracts from H. anthochroum Blaci were determined by simple and multiple antagonism bioassays, and gas phase and agar dilution bioassays respectively. The volatile and semi-volatile metabolites were identified by gas chromatography-mass spectrometry. VOCs from a 5-day H. anthochroum strain Blaci culture caused the inhibition of seed germination, root elongation and seedling respiration on Amaranthus hypochondriacus, Panicum miliaceum, Trifolium pratense and Medicago sativa. In addition, extracts, phenylethyl alcohol and eucalyptol main compounds present in the VOCs and extract displayed a high phytotoxic activity, inhibiting the three physiological processes on the four test plants in a concentration-dependent manner. CONCLUSIONS: The results revealed that H. anthochroum strain Blaci produces a mixture of VOCs. These VOCs showed a strong phytotoxic activity on seed germination, root elongation, and seedling respiration of four plants and slightly affected the growth of phytopathogenic fungi and oomycetes. Also, the culture medium and mycelium extracts of H. anthochroum showed a high phytotoxic activity on the four test plants and, generally, the culture medium extract was more phytotoxic than the mycelium extracts. SIGNIFICANCE AND IMPACT OF THE STUDY: This work firstly reports the phytotoxic activity of volatile and semi-volatile compounds produced by the endophyte H. anthochroum strain Blaci on seed germination, root elongation, and seedling respiration of four different plants; consequently, these compounds could be useful in biocontrol of weeds and plant pathogens.

Antifungal, anti-oomycete and phytotoxic effects of volatile organic compounds from the endophytic fungus Xylaria sp. strain PB3f3 isolated from Haematoxylon brasiletto.

AIMS: To determine the antifungal, anti-oomycete and phytotoxic activity; and chemical composition of the volatile organic compounds (VOCs) produced by endophytic fungus Xylaria sp. PB3f3 isolated from Haematoxylon brasiletto Karst. METHODS AND RESULTS: Bioactivity and chemical composition of the VOCs from Xylaria sp. PB3f3 were established by using simple and multiple antagonism bioassays, and gas chromatography/mass spectrometry, respectively. The results showed that Xylaria sp. PB3f3 inhibited the growth of the oomycetes Pythium aphanidermatum (78·3%), Phytophthora capsici (48·3%), and the fungi Alternaria solani (24·5%) and Fusarium oxysporum (24·2%), in multiple antagonism bioassays. Volatile organic compounds, produced at 20 and 30 days of fungal growth, inhibited root elongation on Amaranthus hypochondriacus (27·6%) and on Solanum lycopersicum (53·2%). Forty VOCs were identified at 10, 20 and 30 days in Xylaria sp. PB3f3 cultures. The compounds with the highest fibre affinity were: 3-methyl-1-butanol and thujopsene, at 10 days of fungal growth; an unidentified amine and 2-methyl-1-butanol at 20 days; and 2-methyl-1-propanol at 30 days. In the gas phase assay method 2-methyl-1-propanol and 2-methyl-1-butanol showed significant inhibitory effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. CONCLUSIONS: Xylaria sp. PB3f3 and its VOCs showed significant phytotoxic effects on root elongation and germination of Am. hypochondriacus and S. lycopersicum. SIGNIFICANCE AND IMPACT OF THE STUDY: The genus Xylaria produces a great variety of secondary metabolites, but, up date, there are no reports of the identification of bioactive volatile compounds. Thus, Xylaria sp. PB3f3 and its VOCs are a possible candidate for the biological control of weeds.

[Epidemiology in inflammatory bowel disease in five areas of Asturias. Spain]. / Epidemiología de la enfermedad inflamaroria intestinal crónica en cinco áreas de Asturias. España.

AIMS: The epidemiologic analysis inflammatory bowel disease (IBD) is a powerful research tool to assess the contribution of environmental factors to its etiology. IBD has been reported to have varying frequencies in different parts of the world, and there seem to be significant differences in the disease pattern and clinical course. The aim of the present study was to assess the disease pattern of IBD in Asturias (Spain). PATIENTS AND METHODS: A descriptive epidemiological population based study, retrospective (1954-1993) and prospective (1994-97), was performed to study 1018 patients found, bigger than 14 years, to have IBD, in five areas of Asturias (Spain) (461.965 inhabitants). RESULTS: During the period of time studied, we diagnosed 1018 IBD [565 ulcerative colitis (55.5%), 415 (40.8%) Crohn's disease and 38(3.7%) indeterminate colitis], with 482 females (47.2%), 536 males (52.8%), and male/female: 1.11. Age at diagnosis were 39.49 +/- 1.08 (95% CI : 38.41 +/- 40.57); (UC: 43.95 +/- 1.47; CD: 33.53 +/- 1.51; IC: 38.26 +/- 5.14. p = 0.000. Age at onset previously at diagnosis for UC: 42.84 +/- 1.34; CD: 30.68 +/- 1.40; IC: 36.74 +/- 4.86 (p = 0.000). Diagnosis criteria: UC: syntomatic 97.34% (p = ns), endoscopy 96.63% (p = 0.000 pathology 90.26% (p = 0.000). CD: radiology 83.61% (p =0.000). Study level in CD: 57.57 (p = 0.0005). Family history: 8.4%. The most frequent involvement at diagnosis of UC was proctitis only, in 13.6%, 269% rectum and sigmoid 26% let colitis, 20% pancolitis, and in CD colon only, in 16.7%, 30.3% terminal ileum, 41.3% ileo-colon of the patients. This also helps to explain the differences in severity, need for surgery, and survival noted between community based studies. CONCLUSIONS: We highlight the uniformity of distribution of the inflammatory bowel disease in relation to types and sex. The high frequency of familial Crohn's disease suggests a genetic predisposition. Highlighting a bigger morbilidad for the Crohn's Disease reflected in the surgical requirements, but however with smaller mortality that in ulcerative colitis.

Epidemiología de la enfermedad inflamatoria intestinal crónica en cinco áreas de Asturias. España / Epidemiology in inflammatory bowel disease in five areas of asturias. Spain

Objetivo: La epidemiología de la enfermedad inflamatoria intestinal crónica (EIIC) es una poderosa herramienta de investigación que contribuye a la evaluación de los factores medioambientales que influyen en su etiología. El objetivo de este estudio es conocer distintos aspectos epidemiológicos de la EIIC en nuestro medio. Pacientes y métodos: Estudio epidemiológico descriptivo, poblacional, multicentrico, retrospectivo entre 1954 y 1993 y prospectivo entre 1994 y 1997. Se incluyen 1018 enfermos mayores de 14 años, diagnosticados de EIIC en 5 áreas del Principado de Asturias (España), con un censo de 461.965 habitantes. Resultados: Del total de 1018 identificados [565 CU (55,5 por ciento) (incluyendo proctitis), 415 EC (40,8 por ciento) y 38 CI (3,7 por ciento)], 482 son mujeres (47,2 por ciento) y 536 varones (52,8 por ciento), con una relación V/M de 1,11. La edad media al diagnóstico es de 39,49 ± 1,08 (IC; 95 por ciento: 38,41 - 40,57), [CU: 43,95 ± 1,47; EC: 33,53 ± 1,51; CI: 38,26 ± 5,14]. p = 0,000. La edad media de inicio de síntomas previo al diagnóstico es 37,66 ± 0,97 (CU: 42,84 ± 1,34; EC: 30,68 ± 1,40; CI: 36,74 ± 4,86 (p = 0,000). El diagnóstico de CU ha sido posible con criterios clínicos en el 97,34 por ciento (p = ns), criterios endoscópicos en el 96,63 por ciento (p = 0,000) y criterios histológicos en el 90,26 por ciento (p = 0,000). En la EC: criterios radiológicos 83,61 por ciento (p = 0,000). El nivel cultural es superior en la EC: 57,57 (p = 0,0005). Asociación familiar del 8,4 por ciento. Extensión: en la CU: proctitis 13,6 por ciento, 26,9 por ciento colitis distal, 26 por ciento colitis izquierda, 6 por ciento colitis extensa y el 20 por ciento pancolitis; En la EC el 30,3 por ciento tienen afectación de íleon terminal, el 16,7 por ciento colon, el 41,3 por ciento colon e intestino, el 11,7 por ciento son intestinales extensas y el 3,7 por ciento tienen afectación gastro-duodenal; En la CI destaca un 39,5 por ciento de afectación discontinua. La media de cirugías necesarias para el control de la enfermedad es de 0,44 ± 6,11, (26,62 por ciento de los enfermos). CU: 0,12 ± 3,33 (9,91 por ciento); EC: 0,91 ± 12,9 (50,36 por ciento), p = 0,000. Tasa de Mortalidad de 47,15 /1000 habitantes (CU: T = 61,94; EC: T = 26,50; CI: T= 0,004) p = 0,046. RMS: 0,467 (CU: 6,14; EC: 2,63; CI: 100). Conclusiones: Este estudio que abarca una importante población de enfermos, pretende aportar nuestros resultados epidemiológicos a la Enfermedad Inflamatoria Intestinal Crónica. Nuestros resultados no difieren substancialmente de los de otras publicaciones. La colitis ulcerosa y la enfermedad de Crohn así como el sexo, se distribuyen uniformemente. La elevada asociación familiar entre estas enfermedades sugiere un origen genético de la EIIC. La enfermedad de Crohn se expresa con mayor morbilidad reflejada en los requerimientos quirúrgicos, pero sin embargo con menor mortalidad que en la colitis ulcerosa (AU)

[Incidence and prevalence of inflammatory bowel disease. Asturian study in 5 areas (EIICEA). Spain]. / Incidencia y prevalencia en enfermedad inflamatoria intestinal crónica. Estudio asturiano en cinco áreas (EIICEA). España.

AIMS: To know and to compare Inflammatory Bowel Disease (IBD) Incidence and Prevalence rates in in five areas of Asturias (Spain). We conducted a prospective epidemiologic study of IBD in the Province of Liege (1 million inhabitants). PATIENT AND METHODS: We conducted a descriptive, populational, collaborative epidemiologic study, retrospective between 1954 and 1993 and prospective between 1994 and 1997. All patients diagnosed according to a standard protocol for case ascertainment and definition of IBD, aged 14 years or more are included, in five areas of Asturias (Spain) (461,965 inhabitants). RESULTS: For the period 1954 to 1997, 1018 IBD have been diagnosed [565 ulcerative colitis (UC) (55.5%), 415 Crohn's disease (CD) (40.8%) and 38 undefined IBD (IC) (3.7%)]; [482 women (47.2%), 536 males (52.8%)]. In the 4 year-prospective period, 306 cases were collected: 176 UC (57.51%), 110 CD (35.94) and 20 IC (6.53%); UC/CD: 1.6. Without appreciable and significant differences between Frequency of illness groups and sexes. IBD incidence rate (per 100,000 per year) (1954-97) is 5.12 (95% CI = 3.05-7.18) (UC: 2.84; CD: 2.08; IC: 0.19; UC/CD 1.36). In the 4 years- prospective study, IBD incidence rate is 16.55 (95% CI = 12.84-20.25), (UC: 9.52; CD: 5.95; IC: 1.08; UC/CD: 1.6). IBD prevalence rate in 1997 is 205.21 (95% CI = 182.14-227.29), (UC: 109.96; CD: 87.45; IC: 7.79). Comparisons have settled down among the studied areas, without finding differences statistically significant. CONCLUSIONS: Inflammatory Bowel Disease incidence and prevalence rates of in our region are homogeneous between the cities investigated and superior than those historically reported in Spanish studies. These results were similar to those observed in European studies.

Estudio multicéntrico. EIICEA. Asturias. Incidencia y prevalencia en enfermedad inflamatoria intestinal crónica. Estudio asturiano en cinco áreas (EIICEA). España / Incidence and prevalence of inflammatory bowel disease in Asturias (Spain)

Objetivo: Conocer y comparar la incidencia y prevalencia de la enfermedad inflamatoria intestinal crónica (EIIC) en 5 áreas del Principado de Asturias (España).Pacientes y métodos: Estudio epidemiológico descriptivo, poblacional, multicéntrico, retrospectivo entre 1954 y 1993 y prospectivo entre 1994 y 1997. Se incluyen todos los enfermos mayores de 14 años, diagnosticados de EIIC según un protocolo estándar para el diagnóstico y definición, en 5 áreas del Principado de Asturias, con un censo de 461.965 habitantes. Resultados: En el periodo de tiempo estudiado, han sido diagnosticados 1018 enfermos con EIIC [565 CU (55,5%), 415 EC (40,8%) y 38 CI (3,7%)]; [482 mujeres (47,2%), 536 varones (52,8%)]. En el periodo de 4 años de estudio prospectivo, se identifican 306 EIIC: 176 CU (57,51%), 110 EC (35,94) y 20 CI (6,53%); CU/EC: 1,6. La frecuencia de aparición de los distintos grupos de enfermedad no presenta diferencias significativas, así como tampoco existen diferencias entre ambos sexos. La tasa de incidencia media anual (1954-97) en EIIC es 5,12 (IC 95% = 3,05 - 7,18) (CU: 2,84; EC: 2,08; CI: 0,19; CU/EC 1,36). En el periodo de tiempo de estudio prospectivo, la tasa de incidencia media anual de la EIIC es 16,55 (IC 95% =12,84 - 20,25), (CU: 9,52; EC: 5,95; CI: 1,08; CU/EC: 1,6). La prevalencia, referida a 1997 para la EIIC es de 205,21 (IC 95% = 182,14227,29), (CU: 109,96; EC: 87,45; CI: 7,79). Se han establecido comparaciones entre las áreas estudiadas, sin encontrar diferencias estadísticamente significativas. Conclusiones: Las tasas brutas de incidencia y de prevalencia de la enfermedad inflamatoria intestinal crónica en nuestro medio son superiores a las históricamente descritas en otras áreas de nuestro país y similares a las publicadas en poblaciones de alta incidencia. No hemos encontrado diferencias significativas entre las cinco áreas que componen el estudio (AU)

The Arabidopsis thaliana ABC protein superfamily, a complete inventory.

We describe the first complete inventory of ATP-binding cassette (ABC) proteins from a multicellular organism, the model plant Arabidopsis thaliana. By the application of several search criteria, Arabidopsis was found to contain a total of 129 open reading frames (ORFs) capable of encoding ABC proteins, of which 103 possessed contiguous transmembrane spans and were identified as putative intrinsic membrane proteins. Fifty-two of the putative intrinsic membrane proteins contained at least two transmembrane domains (TMDs) and two nucleotide-binding folds (NBFs) and could be classified as belonging to one of five subfamilies of full-molecule transporters. The other 51 putative membrane proteins, all of which were half-molecule transporters, fell into five subfamilies. Of the remaining ORFs identified, all of which encoded proteins lacking TMDs, 11 could be classified into three subfamilies. There were no obvious homologs in other organisms for 15 of the ORFs which encoded a heterogeneous group of non-intrinsic ABC proteins (NAPs). Unrooted phylogenetic analyses substantiated the subfamily designations. Notable features of the Arabidopsis ABC superfamily was the presence of a large yeast-like PDR subfamily, and the absence of genes encoding bona fide cystic fibrosis transmembrane conductance regulator (CFTR), sulfonylurea receptor (SUR), and heavy metal tolerance factor 1 (HMT1) homologs. Arabidopsis was unusual in its large allocation of ORFs (a minimum of 0.5%) to members of the ABC protein superfamily.

Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2.

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.

Evidence for posttranscriptional activation of gamma-glutamylcysteine synthetase during plant stress responses.

Glutathione (GSH) is a key component of plant antioxidant defenses. We have sought to determine how the rate-limiting step in GSH biosynthesis, catalyzed by gamma-glutamylcysteine synthase (gammaECS) is regulated in Arabidopsis. Functional complementation of a yeast mutant deficient in this enzyme with an Arabidopsis expression library yielded two cDNAs with sequence identical to the previously described AtgammaECS. Nevertheless, the cellular concentration of GSH in these transformants was only 10% of wild-type concentrations and this was not a result of Cys availability. To explore the possibility that Arabidopsis gammaECS requires additional factors for full catalytic activity, we analyzed the GSH levels and the enzyme activities and transcript levels of both enzymes of the GSH biosynthetic pathway in Arabidopsis suspension cultures subjected to a variety of stresses that raise GSH levels. Our results demonstrate rapid posttranscriptional activation of Arabidopsis gammaECS. The implications of these findings for the mechanisms by which GSH concentrations are regulated during plant-stress responses are discussed.

[Orthopedic shoes and their modifications]. / Calzado ortopédico y sus modificaciones.

The bone structure of the foot, knee and hip and their principle deformities are presented. With this in mind, the orthopedic shoe and its modifications are discussed as an extremely important element in correcting deformities that cause walking alterations, pain, etc.... How the shoes are made, the basic characteristics they should possess, which shoes are appropriate for which age, and problems the wrong shoe type can provoke are also discussed.

Cloning and expression analyses of AtMRP4, a novel MRP-like gene from Arabidopsis thaliana.

In all organisms glutathione-conjugate transporters (GS-X pumps) mediate the detoxification of a number of xenobiotics by removing them from the cytosol. In addition, GS-X pumps appear to play a role in the processing of endogenous compounds. We have isolated a novel genomic clone from Arabidopsis thaliana that encodes a putative GS-X pump, AtMRP4, which is part of a recently defined gene family. The derived amino acid sequence shares high levels of similarity (55-63%) with human, yeast, and other Arabidopsis homologues. The expression of the different members of the AtMRP gene family in Arabidopsis cell suspensions after treatment with chemicals that modify glutathione metabolism (compounds that induce different types of stress and that act as herbicide antidotes- safeners- in monocotyledonous species) revealed that the members of this gene family are differentially regulated.

Inhibition of gibberellin biosynthesis by nitrate in Gibberella fujikuroi.

Gibberellin production in Gibberella fujikuroi starts upon exhaustion of the nitrogen source. To determine the role of nitrate and ammonium in the regulation of gibberellin biosynthesis we have isolated mutants that cannot use nitrate as a nitrogen source. Nitrate inhibited partially the production of gibberellins in mutants devoid of nitrate reductase activity. The inhibition occurred whether nitrate was added before or after the onset of gibberellin production. Addition of tungstate to the wild type mimicked the results with nitrate reductase mutants. We conclude that nitrate inhibits gibberellin biosynthesis by itself, independently of the intracellular signal that conveys nitrogen availability.

Cell proliferation and hair tip growth in the Arabidopsis root are under mechanistically different forms of redox control.

We provide evidence that the tripeptide thiol glutathione (GSH) participates in the regulation of cell division in the apical meristem of Arabidopsis roots. Exogenous application of micromolar concentrations of GSH raised the number of meristematic cells undergoing mitosis, while depletion of GSH had the opposite effect. A role for endogenous GSH in the control of cell proliferation is also provided by mapping of GSH levels in the root meristem using the GSH-specific dye monochlorobimane and confocal laser scanning microscopy. High levels of GSH were associated with the epidermal and cortical initials and markedly lower levels in the quiescent center. The mechanisms controlling cell division could also be triggered by other reducing agents: ascorbic acid and dithiothreitol. Our data also reveal significant plasticity in the relationship between the trichoblast cell length and the hair it subtends in response to alterations in intracellular redox homeostasis. While mechanisms that control trichoblast elongation are influenced by nonspecific redox couples, root hair tip growth has a more specific requirement for sulfhydryl groups. The responses we describe here may represent the extremes of redox control of root plasticity and would allow the root to maintain exploration of the soil under adverse conditions with minimal cell divisions and root hair production or capitalize on a favorable environment by production of numerous long hairs. Redox sensing of the environment and subsequent redox-dependent modulation of growth and development may be crucial components in the strategies plants have evolved for survival in a fluctuating environment.

[An analysis of the reliability of 2 types of enteral nutrition pumps]. / Análisis de la fiabilidad en dos tipos de bombas de nutrición enteral.

The main advantages of infusion pumps are their enhanced accuracy and safety in providing enteral nutrition. We proposed to observe pumps reliability in connection with a variety of factors such as the type of pump used, the administration rate and the energy density of the diets used. An experimental design was prepared with two types of enteral nutrition pumps-VP, volumetric pump, and PP, peristaltic pump. The clinical simulation was done by connecting the enteral nutrition equipment to a graduated dosing system, making hourly measurements of the volume infused. The Reliability Index (RI) used was the ratio between the volume infused by the pump and the regulated dose (Vi/Do). Five volumetric pumps (FLEXIFLO COMPANION) and five peristaltic pumps (FLEXIFLO II) were selected at random from a single commercial outlet. Each was used for twenty-three hours, without interruption, to infuse five types of diet with different energy densities (D1:2 Kcal/ml, D2: 1.5 Kcal/ml, D3: 1.25 Kcal/ml, D4: diet with fibre, and D5: reconstituted powder diet) at three different flow rates (40, 80 and 120 ml/h), to provide fifteen experimental conditions with each pump. Reliability was maintained for both types of pump within the margin of error claimed by the manufacturer (+/- 10%) except in the first hour of infusion (RI = 71.4%). Comparison of the overall RI between the two pump types revealed significant differences, with an RI below the reference range for the VP only during hour 23 (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus niger niaD gene encoding nitrate reductase.

A transformation system for Gibberella fujikuroi based on the Aspergillus niger nitrate reductase gene (niaD) was developed. A strain (designated SG140) carrying a non-reverting niaD mutation (niaD11) was generated by screening mutagenised cells for non-growth on nitrate as sole nitrogen source. Transformation frequencies of 1-2 transformants per microgram DNA were observed when strain SG140 was transformed to nitrate utilisation. Southern blot analyses of niaD+ transformants showed that the vector DNA sequences were integrated into the chromosomal DNA. The results demonstrate that the A. niger niaD gene is expressed in G. fujikuroi.