Evaluation of fire recurrence effect on genetic diversity in maritime pine (Pinus pinaster Ait.) stands using Inter-Simple Sequence Repeat profiles.

The management of maritime pine in fire-prone habitats is a challenging task and fine-scale population genetic analyses are necessary to check if different fire recurrences affect genetic variability. The objective of this study was to assess the effect of fire recurrence on maritime pine genetic diversity using inter-simple sequence repeat markers (ISSR). Three maritime pine (Pinus pinaster Ait.) populations from Northern Portugal were chosen to characterize the genetic variability among populations. In relation to fire recurrence, Seirós population was affected by fire both in 1990 and 2005 whereas Vila Seca-2 population was affected by fire just in 2005. The Vila Seca-1 population has been never affected by fire. Our results showed the highest Nei's genetic diversity (He=0.320), Shannon information index (I=0.474) and polymorphic loci (PPL=87.79%) among samples from twice burned populations (Seirós site). Thus, fire regime plays an important role affecting genetic diversity in the short-term, although not generating maritime pine genetic erosion.

Termómetros de mercurio, aún tóxicos aún presentes / Mercury thermometers, still toxic, still present

Los termómetros de mercurio han sido y son, a pesar de la prohibición de su fabricación, una de las principales fuentes de exposición en la edad pediátrica al mercurio (Hg) elemental en nuestro medio. La toxicidad producida por el Hg elemental depende de la vía de exposición y de la duración de la misma. La exposición a través del tracto digestivo no produce prácticamente toxicidad, pero la inoculación subcutánea o endovenosa y la inhalación puede producir lesiones a nivel local o sistémico. Presentamos el caso clínico de una niña, que presentó inoculación de mercurio líquido en tejido subcutáneo tras la rotura de un termómetro de cristal, produciéndose daño a nivel local con este atonecrosis del tejido. El diagnóstico se realizó mediante estudio radiológico y precisó intervención quirúrgica urgente con escisión en cuña de piel y tejido subcutáneo, guiada con radioscopia. Se descartó la extensión a nivel sistémico, comprobándose la normalidad de los niveles de mercurio en sangre y orina(AU)

Mercury thermometers are and have been, despite their manufacture being banned, one of the main sources of exposure at the paediatric age to elementary mercury (Hg) in our environment. The toxicityproduced by elementary Hg depends on the exposure channel and its length. Exposure through the digestive tract produces hardly any toxicity, but subcutaneous or intravenous inoculation and inhalation of mercury may produce damages at a local or system level. We present the case of a child who showed inoculation of liquid mercury in subcutaneous tissue after a liquid-in-glass thermometer broke. This provoked damages at a local level with steatonecrosis of the tissue. The diagnosis was decided through a radiological test and required urgent surgery with excision of skin and subcutaneous tissue, guided by radioscopy. Any spread at a system level was discarded. The levels of mercury in the bloodstream and in the urine were regular (AU)

[Mercury thermometers, still toxic, still present]. / Termómetros de mercurio, aún tóxicos aún presentes.

Mercury thermometers are and have been, despite their manufacture being banned, one of the main sources of exposure at the paediatric age to elementary mercury (Hg) in our environment. The toxicity produced by elementary Hg depends on the exposure channel and its length. Exposure through the digestive tract produces hardly any toxicity, but subcutaneous or intravenous inoculation and inhalation of mercury may produce damages at a local or system level. We present the case of a child who showed inoculation of liquid mercury in subcutaneous tissue after a liquid-in-glass thermometer broke. This provoked damages at a local level with steatonecrosis of the tissue. The diagnosis was decided through a radiological test and required urgent surgery with excision of skin and subcutaneous tissue, guided by radioscopy. Any spread at a system level was discarded. The levels of mercury in the bloodstream and in the urine were regular.

Molecular cloning and characterisation of a pathogenesis-related protein CsPR10 from Crocus sativus.

Plants have developed many mechanisms to protect themselves against most potential microbial pathogens and diseases. Among these mechanisms, pathogenesis-related proteins are produced as part of the active defence to prevent attack. In this study, a full-length cDNA encoding the CsPR10 protein was identified in fresh saffron stigmas (Crocus sativus). The deduced amino acid sequence from the nucleotide sequence of the coding region showed homology with PR10 proteins. The clone expressed as a protein in fusion with a GST tag produced a 47-kDa protein in E. coli. CsPR10 had ribonuclease activity, with features common to class II-type ribonucleases; its specific activity was quantified as 68.8 U·mg(-1) protein, thus falling within the range of most PR10 proteins exhibiting RNase activity. Antifungal activity of CsPR10 was assayed against Verticillium dahliae, Penicillium sp. and Fusarium oxysporum. CsPR10 inhibited only F. oxysporum growth, and antifungal potency was reflected in a IC(50) of 8.3 µm. Expression analysis showed the presence of high transcript levels in anther and tepal tissues, low levels in stigmas and roots, and no signal detected in leaves. This protein seems to be involved in the active defence response through activation of the jasmonic acid pathway.

Involvement of lipid transfer proteins in saffron hypersensitivity: molecular cloning of the potential allergens.

BACKGROUND: Lipid transfer proteins (LTPs) are relevant allergens, and have recently been proposed as model plant allergens from fruit, vegetables, seeds, and pollens. However, no LTP spice allergen has been characterized to date. OBJECTIVES: To identify and isolate saffron LTPs and to explore their relevance in saffron allergy. METHODS: Six patients with rhinitis and positive skin prick test (SPT) results to saffron extract were selected. Two recombinant LTPs from saffron were isolated, cloned into pPIC9 plasmid, and produced in Pichia pastoris. Immunoglobulin (Ig) E immunodetection and enzyme-linked immunosorbent assays were performed with the 2 purified allergens and with the major peach allergen Pru p 3. RESULTS: Full cDNA corresponding to 2 saffron LTP variants was isolated and expressed in P pastoris. The molecular weight of rCro s 3.01 and rCro s 3.02 was 9.15 kDa and 9.55 kDa, respectively. The sequences obtained had a 47% identity with each other and 51% and 43% with Pru p 3. Both proteins were recognized by anti-Pru p 3 antibodies. Specific IgE to the purified allergens was found in 50% of patients for rCro s 3.01 and 33% for rCro s 3.02 and Pru p 3 in the saffron-allergic patients. CONCLUSIONS: Our results indicated that rCro s 3.01 and rCro s 3.02 are minor allergens of saffron, at least in the study patients. To our knowledge, this is the first report on the implication of LTPs in spice allergy.

Tratamiento del niño quemado en Atención Primaria / Treatment of burns in children in Primary Care

Las quemaduras en la infancia, a pesar de las campañas preventivas, continúan siendo un accidente frecuente. Los niños pequeños son la población más afectada. La mayoría no son graves y sólo precisan atención ambulatoria, pero, en ocasiones, pueden producir una importante morbimortalidad. Actualmente continúan siendo la tercera causa de muerte en la infancia. La causa más frecuente suelen ser las escaldaduras y éstas se producen habitualmente en el domicilio y, en muchas ocasiones, en presencia de algún adulto. El manejo inicial correcto y una valoración adecuada de la gravedad son importantes en el pronóstico. La mayoría de las quemaduras son evitables. Divulgar e insistir en las medidas preventivas continúa siendo fundamental para el descenso de este problema. Por su accesibilidad, el pediatra de Atención Primaria debe tener un papel destacado en este objetivo (AU)

In spite of the preventive campaigns, paediatric burns are still a frequent accident. Small children are the most affected population. Although the majority of burns are not serious and only need ambulatory care, a reduced number can produce great morbidity and even mortality. Currently burns continue being the third cause of death in paediatrics. Burns are most frequently caused by hot liquids and they often happened at home in the presence of some adult people. A correct initial management and an adequate appraisal of the severity are very important to set the right treatment needed and to predict the outcome. Almost all burns are avoidable. We have to face the task of spread educational and preventive measures to take out this problem. The paediatrician of Primary Care is, by his own nature, the main actor in this important target (AU)

Sensitivity of different ecotypes and mutants of Arabidopsis thaliana toward the bacterial elicitor flagellin correlates with the presence of receptor-binding sites.

Flagellin, the main building block of the bacterial flagellum, acts as potent elicitor of defense responses in different plant species. Genetic analysis in Arabidopsis thaliana identified two distinct loci, termed FLS1 and FLS2, that are essential for perception of flagellin-derived elicitors. FLS2 was found to encode a leucine-rich repeat transmembrane receptor-like kinase with similarities to Toll-like receptors involved in the innate immune system of mammals and insects. Here we used a radiolabeled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, to probe the interaction of flagellin with its receptor in A. thaliana. The high affinity binding site detected in intact cells and membrane preparations exhibited specificity for flagellin-derived peptides with biological activity as agonists or antagonists of the elicitor responses. Specific binding activity was measurable in all ecotypes of A. thaliana that show sensitivity to flagellin but was barely detectable in the flagellin-insensitive ecotype Ws-0 affected in FLS1. A strongly impaired binding of flagellin was observed also in several independent flagellin-insensitive mutants isolated from the flagellin-sensitive ecotype La-er. In particular, no binding was found in plants carrying a mutation in the LRR domain of FLS2. These data indicate that the formation of functional receptor-binding sites depends on genes encoded by both loci, FLS1 and FLS2. The tight correlation between the presence of the binding site and elicitor response provides strong evidence that this binding site acts as the physiological receptor of flagellin.

Both the extracellular leucine-rich repeat domain and the kinase activity of FSL2 are required for flagellin binding and signaling in Arabidopsis.

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.

FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis.

Flagellin, the main protein of the bacterial flagella, elicits defence responses and alters growth in Arabidopsis seedlings. Previously, we identified the FLS1 locus, which confers flagellin insensitivity in Ws-0. To identify additional components involved in flagellin perception, we screened for flagellin insensitivity mutants in the flagellin-sensitive accession La-er. Here, we describe the identification of a new locus, FLS2, by a map-based strategy. The FLS2 gene is ubiquitously expressed and encodes a putative receptor kinase. FLS2 shares structural and functional homologies with known plant resistance genes and with components involved in the innate immune system of mammals and insects.

A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana.

Peptides corresponding to the most conserved domain of eubacterial flagellin act as potent elicitors in cells of different plant species. In intact Arabidposis thaliana seedlings these peptides (flg22 and flg15) caused callose deposition, induction of genes coding for pathogenesis-related proteins and a strong inhibition of growth. Half-maximal growth inhibition occurred at peptide concentrations of approximately 100 nM. In contrast, peptides representing the corresponding flagellin domains of the plant-associated bacteria A. tumefaciens and R. meliloti were inactive even at concentrations of 10 microM. With the exception of Ws-0, all ecotypes of A. thaliana tested were sensitive to flg22. Crosses of Ws-0 with the sensitive ecotypes Col-0 and La-er, respectively, resulted in sensitive F1 seedlings. In the F2 generation of both crosses, sensitivity segregated as a single trait with markers of chromosome 5 and a ratio of 3:1. Dominance of the locus sensing flagellin, termed FLS-1, suggests that it encodes an element which is important for the perception of the flagellin signal.

Differential expression of the S-adenosyl-L-methionine synthase genes during pea development.

Two genes coding for S-adenosyl-l-methionine synthase (SAMS, EC 2.5. 1.6) were previously isolated from pea (Pisum sativum) ovaries. Both SAMS genes were highly homologous throughout their coding regions but showed a certain degree of sequence divergence within the 5' and the 3' untranslated regions. These regions have been used as gene-specific probes to analyze the differential expression of SAMS1 and SAMS2 genes in pea plants. The ribonuclease protection assay revealed different expression patterns for each individual gene. SAMS1 was strongly expressed in nearly all tissues, especially in roots. SAMS2 expression was weaker, reaching its highest level at the apex. Following pollination, SAMS1 was specifically up-regulated, whereas SAMS2 was expressed constitutively. The up-regulation of SAMS1 during ovary development was also observed in unpollinated ovaries treated with auxins. In unpollinated ovaries an increase in SAMS1 expression was observed as a consequence of ethylene production associated with the emasculation process. In senescing ovaries both SAMS1 and SAMS2 genes showed increased expression. Ethylene treatment of unpollinated ovaries led to an increase in the SAMS1 mRNA level. However, SAMS2 expression remained unchangeable after ethylene treatment, indicating that SAMS2 induction during ovary senescence was not ethylene dependent. SAMS mRNAs were localized by in situ hybridization at the endocarp of developing fruits and in the ovules of senescing ovaries. Our results indicate that the transcriptional regulation of SAMS genes is developmentally controlled in a specific way for each gene.

Hormonal regulation of S-adenosylmethionine synthase transcripts in pea ovaries.

Two cDNA clones coding for S-adenosyl-L-methionine synthase (SAMs, EC 2.5.1.6) have been isolated from a cDNA library of gibberellic acid-treated unpollinated pea ovaries. Both cDNAs were sequenced showing a high degree of identity but coding for different SAMs polypeptides. The presence of two SAMs genes in pea was further confirmed by Southern analysis. Expression of the SAMs genes in the pea plant was found at different levels in vegetative and reproductive tissues. We characterized the expression levels of SAMs genes during the development or senescence of pea ovaries. Northern analysis showed that transcription of SAMs genes in parthenocarpic fruits was upregulated by auxins in the same manner as in fruits from pollinated ovaries. In both pollinated and 2,4-dichlorophenoxyacetic acid-treated ovaries, and benzyladenine, although able to induce parthenocarpic development, did not affect SAMs mRNA levels. These data are consistent with an active participation of auxins in the upregulation of SAMs during fruit setting in pea and suggest that, at the molecular level, parthenocarpic development of pea ovaries is different for gibberellin- and cytokinin-treated ovaries than for auxin-induced parthenocarpic biosynthesis since treatment of the ovaries with aminoethoxyvinylglycine resulted in a delay of senescence and prevention of SAMs mRNA accumulation. A possible mechanism for hormonal regulation of SAMs during ovary development is discussed.