First Report of Heterobasidion annosum on Pinus pinaster in Spain.

The basidiomycete Heterobasidion annosum (Fr.) Bref. (=Fomes annosus (Fr.) Cooke), one of the most important pathogens in coniferous forests in Europe, Asia, and North America, causes root and butt rot. H. annosum was first recorded on Pinus pinaster Ait. (commonly known as Maritime pine) in France and Great Britain in 1961 (4) and Portugal in 1986 (2). P. pinaster is the most widespread conifer in Spain, with more than 700,000 and 600,000 ha in pure and mixed stands, respectively. Over the last few years, P. pinaster decline was observed in several stands in the center of the Iberian Peninsula. Unusual crown transparency, small needles, foliage discoloration, and early tree death are characteristic decline symptoms associated with the high mortality rate on this species. In June of 2010, 11 trees (40 to 60 years old) with a different degree of decline were felled in two zones (42°2'41″N, 3°18'14″W, elevation 1,096 m and 41°55'40″N, 3°12'3″W, elevation 1,128 m) and cut into sections (stump height, breast height, and near the top). Wood slices were removed from each section and taken to the laboratory. Samples were placed in moist chambers with optimal conditions of humidity and temperature to enhance pathogen growth. After 20 days of incubation in darkness at 25°C, H. annosum (anamorph Spiniger meineckellum [A. Olson] Stalpers) occurred on most of these slices. Conidiophores with subglobose to pyriform conidia (5.8 × 4.2 µm) were observed with a compound microscope. The fungus was isolated to extract DNA by disruption of the mycelium followed by washes with phenol/chloroform/isoamyl alcohol solution (25:24:1). DNA was precipitated with 20% polyethylene glycol solution. PCR was carried out according to the instructions of the manufacturer of Dynazyme II DNA polymerase (Finnzymes Ltd, Espoo, Finland) with ITS primers, 1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and 4 (5'-TCCTCCGCTTATTGATATGC-3'). After DNA purification, samples were sequenced (SECUGEN, Madrid, Spain) and aligned and corrected with Geneious Pro 5.3 to obtain the consensus sequences. Resulting DNA sequences of two isolates were deposited in GenBank (Nos. FR850494 and FR850495), and compared with a Blastn search at GenBank showing 100% identity and 100% coverage with H. annosum sensu stricto, former ISG-P (intersterility group of pines). For pathogenicity tests, 10 seedlings (2 year old) were inoculated with autoclaved P. pinaster wood chips colonized by H. annosum, and 10 control seedlings were inoculated with noncolonized wood chips. Inoculums were prepared by growing H. annosum on 4-mm-diameter wood chips placed on potato dextrose agar media for 3 weeks. The wood chips were put inside an oblique incision made at 6 cm above the soil line and wrapped with Parafilm. After 8 weeks in a growth chamber at 22.5°C with a 14-h photoperiod, the inoculated seedlings showed typical symptoms and 3 seedlings of 10 were dead. H. annosum was previously recorded on P. sylvestris in central Spain (1), causing needle drop, swelling at the stump height, and presence of dead trees by circular areas. This pathogen was also reported on P. nigra in northeastern Spain, associated with defoliation and mortality (3). To our knowledge, this is the first record of H. annosum on P. pinaster in Spain. References: (1) J. Benito-Martínez. An. Jardín Bot. Madrid 3:23, 1943. (2) N. Neves et al. EPPO Bull. 16:505, 1986. (3) J. Oliva et al. Bol. Sanidad Vegetal. Plagas. 34:415, 2008. (4) P. Spaulding. US Dep. Agric. Agric. Handb. 197:100, 1961.

Graded contrast echocardiography in pulmonary arteriovenous malformations.

To compare the results of transthoracic contrast echocardiography (TTCE) adding a grading scale with the results of thoracic computed tomography (CT) in order to optimise the use of both techniques. 95 patients with hereditary haemorrhagic telangiectasia (HHT) were examined with TTCE and thoracic CT to detect pulmonary arteriovenous malformations (PAVMs). According to previous studies, TTCE was divided into a four grade scale depending on the degree of opacification of the left ventricle after the administration of a contrast agent. Of the 95 patients (50.5% female; mean age 46 yrs), none with normal or grade 1 TTCE had detectable PAVMs on thoracic CT. Shunts of grades 2, 3 and 4 were associated with PAVMs according to thoracic CT in 25, 80, and 100% of the cases. There was a statistically significant association between the TTCE grade and the detection of a PAVM by thoracic CT. There were also statistically significant associations between TTCE grade and the cardiac cycle when the contrast was first visible in the left atrium, and size of the feeding artery. Graded TTCE and timing of left atrium opacification may be useful techniques in selecting HHT patients for PAVM screening with thoracic CT scans.

The ß2-adrenergic receptor antagonist ICI-118,551 blocks the constitutively activated HIF signalling in hemangioblastomas from von Hippel-Lindau disease.

One of the major consequences of the lack of a functional VHL protein in von Hippel-Lindau disease, a rare cancer, is the constitutive activation of the HIF pathway. This activation ends up in the generation of Central Nervous System (CNS) Hemangioblastomas among other tumours along the lifespan of the patient. Nowadays, only surgery has been proven efficient as therapy since the systemic attempts have failed. Propranolol, a non-specific ß1-and ß2-adrenergic receptor antagonist, was recently designated as the first therapeutic (orphan) drug for VHL disease. Nevertheless, its ß1 affinity provokes the decrease in blood pressure, being not recommended for low or regular blood pressure VHL patients. In order to overcome the ß1-drawback, the properties of a high specific ß2-adrenergic receptor blocker named ICI-118,551 have been studied. ICI-118,551 was able to decrease Hemangioblastomas cell viability in a specific manner, by triggering apoptosis. Moreover, ICI-118,551 also impaired the nuclear internalization of HIF-1α in Hemangioblastomas and hypoxic primary endothelial cells, reducing significantly the activation of HIF-target genes and halting the tumour-related angiogenic processes. In this work, we demonstrate the therapeutical properties of ICI-118,551 in VHL-derived CNS-Hemangioblastoma primary cultures, becoming a promising drug for VHL disease and other HIF-related diseases.

A mindfulness training program based on brief practices (M-PBI) to reduce stress in the workplace: a randomised controlled pilot study.

Work stress is a major contributor to absenteeism and reduced work productivity. A randomised and controlled study in employee-volunteers (with Perceived Stress Scale [PSS-14]>22) was performed to assess a mindfulness program based on brief integrated mindfulness practices (M-PBI) with the aim of reducing stress in the workplace. The PSS-14 of the employees before and after 8-weeks M-PBI program, as well as after a 20-week follow-up, was assessed (primary endpoint). The employees also carried the following questionnaires (secondary endpoints): Five Facet Mindfulness Questionnaire (FFMQ), Self-Compassion Scale (SCS), Experiences Questionnaire-Decentering (EQ-D), and Maslach Burnout Inventory-General Survey (MBI-GS). Heart Rate Variability (HRV) was measured during each session in a subgroup of employees (n = 10) of the interventional group randomly selected. A total of 40 employees (77.5% female median [SD] age of 36.6 [5.6] years) took part in this study: 21 and 19 in the intervention and control group, respectively. No differences in baseline characteristics were encountered between the groups. Results show a significant decrease in stress and increase in mindfulness over time in the intervention group (PSS-14 and FFMQ; p < 0.05 both). Additionally, an improvement in decentering (EQ-D), self-compassion (SCS) and burnout (MBI-GS) were also observed compared to the control group (p < 0.05 in all). HRV measurement also showed an improvement. In conclusion, a brief practices, 8-weeks M-BIP program is an effective tool to quickly reduce stress and improve well-being in a workplace.

Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange.

ABSTRACT Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), a noncachexia variant of Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV) were co-inoculated as two-, three-, four-, and five-viroid mixtures to Clementine trees grafted on trifoliate orange to evaluate their effect on symptom expression, tree growth, and fruit yield. Most trees infected with CEVd-containing viroid mixtures developed exocortis scaling symptoms, as did CEVd alone, whereas most trees infected with HSVd- or CVd-IV-containing mixtures developed bark-cracking symptoms. Trees infected with mixtures containing both CEVd and CVd-IV revealed the existence of antagonism between these two viroids in terms of the expected bark-scaling and cracking symptoms. Synergistic interactions also were identified in trees infected with certain viroid combinations that, in spite of lacking CEVd, expressed exocortis-like scaling symptoms. Viroid interactions also affected the expected response of trees in terms of vegetative growth and fruit yield. Trees infected with viroid combinations containing CEVd or CVd-III were smaller and produced less fruit than trees infected with mixtures not containing these viroids. Viroid interactions on scion circumference and cumulative fruit yield, in terms of additivity of their effects, were statistically confirmed using a factorial analysis of variance model with two mean estimation approaches. In single-viroid infections, CEVd, CVd-III, and, to a lesser extent, CBLVd consistently and significantly reduced tree size and fruit yield. Conversely, HSVd and CVd-IV slightly increased fruit yield and reduced scion circumference. Rare and not consistent significant interactions were detected with the five-, four-, and three-viroid combinations. Antagonistic interactions between CEVd and CVd-III or CBLVd and CVd-III were revealed over the years with consistent significance. The antagonistic interaction between CEVd and CVd-IV was highly significant over the years when additional viroids were present; however, this antagonism appeared much later in the case of an exclusive interaction. HSVd and CVd-IV showed a consistent and significant synergistic interaction on yield only when both viroids were exclusively present. These results demonstrate antagonistic or synergistic relationships between citrus viroids depending on the viroid mixtures present in the host.

Cloning and characterization of the scute (sc) gene of Drosophila subobscura.

The scute (sc) gene, a member of the achaete-scute complex of Drosophila melanogaster, has dual functions: sisterless (sis-b) function required for sex determination and dosage compensation and scute function, which is involved in neurogenesis. The sc homologue of D. subobscura was cloned. It lacks introns and encodes a single 1.7-kb transcript slightly larger than that of D. melanogaster (1.6 kb). The sc protein of D. subobscura is slightly larger than that of D. melanogaster (382 vs. 345 amino acids). Sequence comparisons between both species show the Sc protein to have a highly conserved bHLH domain. Outside this domain, amino acid replacements are not randomly distributed. Two additional conserved domains, of 20 and 36 amino acids, are present near the C-terminal end. They may represent domains confering specificity upon the Sc protein with respect to other proteins of the achaete-scute complex. In its 3' untranslated region, Sc RNA contains uridine stretches, putative Sxl protein DNA-binding sites. The D. subobscura Sc protein can cooperate with other D. melanogaster bHLH proteins because D. subobscura sc supplies sis-b function when introduced into D. melanogaster transgenic flies mutant for sc.

Atorvastatin-induced endothelial nitric oxide synthase expression in endothelial cells is mediated by endoglin.

Endoglin, a transforming growth factor ß (TGF-ß) receptor type III, is co-expressed with endothelial nitric oxide synthase (eNOS) in aortic endothelium in atherosclerotic plaques of mice. Interestingly, atorvastatin (ATV) is able to increase both endoglin and eNOS expression and reduce plaque size beyond its lipid lowering effects but by unknown mechanisms. We hypothesized whether inflammation modulates ATV-dependent induction of endoglin and eNOS expression in vitro in endothelial cells and whether ATV-induced eNOS expression is regulated via endoglin. After treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α, endoglin and eNOS protein expression was reduced, concomitantly with increased levels of cell surface VCAM-1 and soluble endoglin, as determined by flow cytometry, Western blot and ELISA analyses. By contrast, ATV treatment increased endoglin and eNOS protein expression, while preventing TNF-α-mediated downregulation of endoglin and eNOS protein levels. Moreover, suppression of endoglin using small interfering RNA (siRNA), but not inhibition of TGF-ß signaling with SB431542, abrogated ATV-induced eNOS expression. These results suggest that ATV treatment prevents inflammation-reduced endoglin and eNOS expression in endothelial cells and that ATV-induced eNOS expression strongly depends on the proper expression of endoglin in HUVECs. Possible implications of these findings might be reflected in pathological conditions characterized by reduced expression of endoglin and eNOS as for example in hereditary hemorrhagic telangiectasia or in other endothelial dysfunctions.

Molecular characterization of the C-3 DNA puff gene of Rhynchosciara americana.

We have mapped a region of about 33 kb which includes the transcription unit of the C-3 DNA puff gene of Rhynchosciara americana. The C-3 TU and a region extending approximately 800 bp upstream of the C-3 promoter were characterized. The TU is composed of three exons and produces a 1.1-kb mRNA whose level in salivary glands increases with the expansion of the C-3 puff. The C-3 messenger appears to undergo rapid deadenylation resulting in an RNA of about 0.95 kb which can still be observed in gland cells 15 h after the puff has regressed. The 1.1-kb mRNA codes for a 32.4-kDa, predominantly alpha-helical polypeptide with three conserved parallel coiled-coil stretches. The aa composition and structure of this polypeptide suggests that it is secreted and contributes to the formation of the cocoon in which the larvae pupate. The region upstream of the promoter contains several A-rich sequences with similarity to the ACS of yeast which might have a role in the initiation of replication/amplification.

The pesticide malathion induces alterations in actin cytoskeleton and in cell adhesion of cultured breast carcinoma cells.

We have studied the effects of the organophosphorous pesticide malathion on cell viability, actin cytoskeleton, cell adhesion complex E-cadherin/beta-catenin, and Rho and Rac1 GTPases from the human mammary carcinoma cell line MCF-7. Malathion induced cell lethality, determined by the MTT assay, depending on the treatment conditions. Cells incubated with low concentrations of malathion, 16-32 microg/ml, showed high survival rates (>95%) at any evaluated time (1-5 days), whereas complete cell lethality was found using 512 microg/ml and 5 days of treatment. Deep morphological changes were induced with high doses of 64 and 128 microg/ml, and long incubation time (5 days); cells showed perinuclear vacuoles, rounding, shrinkage, and a gradual loss of adhesion. These changes were related to a decrease in the expression of the adhesion molecules, E-cadherin and beta-catenin, and to the distribution and reactivity of actin microfilaments to TRITC-phalloidin. Disruption of microfilaments, accompanied by the collapse of actin to perinuclear region, were characteristic of cells with loss of adhesion. At lower concentrations, some cells presented deformations on the plasma membrane as lamellipodia-like structures, which were particularly evident from 32 to 128 microg/ml. Conversely, we observed an increase in the expression of Rho and Rac1 GTPases, modulators of actin cytoskeleton and cell adhesion.

Citrus Viroids: Symptom Expression and Effect on Vegetative Growth and Yield of Clementine Trees Grafted on Trifoliate Orange.

Citrus are natural hosts of five viroid species: Citrus exocortis viroid (CEVd), Citrus bent leaf viroid (CBLVd), Hop stunt viroid (HSVd), Citrus viroid III (CVd-III), and Citrus viroid IV (CVd-IV). CEVd and specific sequence variants of HSVd are the causal agents of the wellknown diseases of citrus, exocortis and cachexia. Other viroids have been found to induce different degrees of stunting. Since commercial citrus trees are commonly infected with mixtures of these viroids, only limited information is available on their effect in species other than Etrog citron. A field assay was conducted to establish the effect of each viroid on Commune clementine trees grafted on Pomeroy trifoliate orange. Infected trees were periodically monitored over a 12-year period (1990 to 2002) for symptom expression, growth, and fruit yield. Only CEVd caused bark scaling on the trifoliate orange rootstock and marked dwarfing, both characteristic of exocortis disease as initially described. In addition, very conspicuous bumps were observed in the wood of the rootstock after removing the bark. Only those HSVd variants, previously characterized as pathogenic in several cachexia-sensitive species, induced pits and gum deposits characteristic of this disease in the clementine scion. Bark cracking symptoms on the trifoliate orange rootstock were also observed. They were associated with CVd-IV, HSVd, or CEVd infection, but in the latter, they were only clearly observed in trees that showed mild scaling. Other abnormalities (deep pits, crests, and gummy pits) were not associated with viroid infection. No specific symptoms resulted from infection with CBLVd and CVd-III. HSVd, CVd-IV, and CBLVd had little or no effect in growth and yield, whereas CEVd and CVd-III caused a significant reduction of growth and yield, which became more pronounced over time with CEVd infection. Yield reduction was associated mainly with loss of production of large fruits. In general, there was a good correlation between reduction in vegetative growth and yield.

Organophosphorous pesticides in breast cancer progression.

Environmental substances may be involved in the etiology of breast cancers. Many studies have found an association between cancer in humans and exposure to agricultural pesticides. Organophosphorous pesticides have been used to control mosquito plagues. Parathion and malathion, organophosphorous pesticides are cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. Their primary target of action in insects is the nervous system whereby they inhibit the enzyme acetylcholinesterase at synaptic junction. Atropine is a parasympatholytic alkaloid used as an antidote to acetylcholinesterase inhibitors. We have established an experimental breast cancer model, where epithelial cells in the rat mammary gland underwent a stepwise transformation into malignant cells by exposure to pesticides (Cabello et al, 2001). The aim of this work was to examine whether pesticides were able to induce progression of malignant transformation of a human breast epithelial cell line, MCF7. These results showed that parathion and malathion increased PCNA and induced mutant p53 protein expression of MCF7 cells in comparison to controls and atropine inhibited such action. These results indicated that organophosphorous pesticides can induce more changes in this malignant breast cell line, inducing another step in the progression of the transformation process and atropine on the other hand inhibited the effect of such substances.

The autobiographical group: a tool for the reconstruction of past life experience with the aged.

From a personal construct point of view, writing autobiographical texts becomes a relevant therapeutic ingredient for elderly individuals. If conducted in a context of a group, as Birren proposes [1, 2]; it promotes self-awareness, self-disclosure, and the capacity of generating alternative views of life's experiences. In a group of elderly volunteers from a recreational society of Barcelona (Spain), the guided autobiography method was used to foster the reconstruction of the participant's past life experiences. The degree of reconstruction was assessed through a design that included the administration of a repertory grid at the initial and tenth (last) session. A parallel assessment was applied to a control group of participants with similar demographic characteristics. An adaptation of the method proposed by Feixas for the analysis of autobiographical texts was used to assess the assigned writings of the participants for each session. Results show a significant and gradual change in the construing system of those participants in the autobiographical group. Thus, the distance of the elements self-ideal/self and self-ideal/others significantly decreased in comparison to the control group. It is suggested that the guided autobiography is an adequate therapeutic tool to promote the reconstruction of experience in aged individuals.

[Effect of beta-ecdysterone in competitive cultures of Drosophila melanogaster]. / Efecto de la beta-ecdisterona en cultivos en competencia de Drosophila melanogaster.

High levels of beta-ecdysterone (more than 1 microgram/ml) have been shown to decrease larva-adult viability and to speed up developmental times when supplied to the media in crowding cultures. The highest doses (100 micrograms/ml) of ecdysterone suppresses almost completely the phenomenon of larval stop in the third instar of development, first reported by our laboratory in overcrowded situations. Thus, one may deduce that stopped larvae could have low levels of ecdysone, and perhaps these are the ultimate physiological cause of their arrested development before the critical larva-pupa molt.

The Balbiani ring 6 induction in Chironomus.

Balbiani ring (BR) genes in polytene chromosomes of Chironomus salivary glands code for secretory proteins of the sp-I family, 10(6) D. They are used by the aquatic larva to spin a housing and feeding tube. The expression of the BR gene family undergoes correlated changes depending on the environment. In the presence of certain sugars, ethanol or glycerol the normally most active BR2 regresses and its products disappear. There is a parallel induction of a new BR, BR6, and a new sp-I protein. The change seems to represent an adaptative response to phosphate depletion in the larval haemolymph produced by the inducing agents. The BR2 (and BR1) products are heavily phosphorylated and the BR6 product non-phosphorylated. One of the BR2-coded sp-I proteins is cleaved off close to the C-terminus and material with properties expected of the resulting polypeptide can be recovered in the nuclei, accumulating in the BR. This might represent a feed-back signal from translation to transcription.

The C-terminal DNA-binding domain of Chironomus BR gene products shows preferential affinity for (dA.dT)-rich sequences.

Balbiani ring genes (BRs), the most active loci in the polytene chromosomes of the salivary gland of the midge Chironomus (Diptera), code for secretory giant peptides (the sp-I family). Evidence previously reported indicated that the conserved C-terminal region of proteins of the sp-I family had DNA-binding properties (assayed with sp-Ia), and one such region, derived from BR2.2, which codes for the product sp-Ib, might occur as a stable independent peptide, being transferred to the nucleus where it is detectable in the large BRs (BR1 and BR2), among other structures, by immunostaining. Here, we show that the C-terminal portion of one of the BR gene products, expressed as a glutathione-S-transferase fusion protein shows preferential affinity for A.T-rich sequences and binds with varying affinity to restriction fragments of the A.T-rich BR1 promoter. The binding was inhibited by distamycin, suggesting that the interaction involves the minor groove of the DNA. Analysis of the promoter fragments by gel electrophoresis indicated that most appeared to present a conspicuous bend, as deduced from their anomalous electrophoretic mobilities. Furthermore, the affinity of the C-terminal domain for the different promoter fragments appeared to correlate with the degree of bending. Thus, the C-terminal domain might play a role in controlling gene expression by binding to A.T-rich sequences, including those of the BR genes.

Functional analysis of the 3'-terminal part of the Balbiani ring 2.2 gene by interspecies sequence comparison.

An interspecies comparison was made between the 3' ends of Balbiani ring genes from Chironomus. The comparison was focused on the BR2.2 gene, and a part at the 3' end from Chironomus pallidivittatus (which included also a segment of the gene core) was cloned. Its sequence, and other previously published BR sequences from this species and from Chironomus tentans were used in the analysis. The 3' parts of these repetitive genes can be divided into a region belonging to the core of the genes followed by a terminal region. In the core region the repeats (each of which consists of a constant part and a subrepeated part) are highly similar and the constant parts show little interspecies differentiation. Furthermore, the two parts of the repeats are units in an evolutionary and probably also functional sense. The terminal region contains modified constant units, usually isolated between acidic so-called cys regions, the whole arrangement lying upstream of an intron toward a 3'-terminal exon. Most of the modified constant units are mosaics in rates of evolution with stable outer quarters bordering to equally stable cys regions and a central half with a very high rate of evolution. One of the terminal units, present only in the BR2.2 gene and second from the end, differs distinctly not only from corresponding core units but also from other terminal units in the three normally active BR genes. It lies upstream of all cys regions and is evolutionarily conserved over most of its length. Furthermore, two-dimensional protein structure prediction does not exclude an endoproteolytic cleavage site in this unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Subrepeats within the BR1 beta repeat unit in Chironomus pallidivittatus can be classified into different types depending on codon usage.

A new type of repeat unit was isolated from Balbiani ring 1 of Chironomus pallidivittatus and designated BR1 beta repeat. It consists of a constant and a subrepeated part, like previously described units belonging to the core blocks of the BR genes. The subrepeated part contains 10-codon subrepeats with an arrangement similar to the subrepeats of the previously described BR2 beta gene. The present unit differs from earlier reported core units firstly in a much lower number of copies (about 15) per genome, which are tandemly arranged. Secondly, the number of subrepeats per BR1 beta repeat unit can show great variations. On the basis of the pattern of codon usage, three types of subrepeats can be distinguished. One type lies 5'-proximal in the subrepeat array and consists of variable numbers of subrepeats almost identical at the nucleotide level. The last complete subrepeat represents another type, with consistent differences in codon usage as compared to subrepeats of the proximal type. Finally, there is an intermediate type represented by the subrepeat preceding the distal one. Here, codon characteristics from proximal and distal subrepeats are mixed in a patchy and irregular way. The evolution of the arrays can be understood either as being the result of subrepeat formation in two steps (occurring before and after amplification of whole repeat units) or as the result of a continuous process in which there is evidence for participation of gene conversion.