176 research outputs found
Isolation and functional characterization of a cotton ubiquitination-related promoter and 5'UTR that drives high levels of expression in root and flower tissues
<p>Abstract</p> <p>Background</p> <p>Cotton (<it>Gossypium </it>spp.) is an important crop worldwide that provides raw material to 40% of the textile fiber industry. Important traits have been studied aiming the development of genetically modified crops including resistance to insect and diseases, and tolerance to drought, cold and herbicide. Therefore, the characterization of promoters and regulatory regions is also important to achieve high gene expression and/or a specific expression pattern. Commonly, genes involved in ubiquitination pathways are highly and differentially expressed. In this study, we analyzed the expression of a cotton ubiquitin-conjugating enzyme (E2) family member with no previous characterization.</p> <p>Results</p> <p>Nucleotide analysis revealed high identity with cotton <it>E2 </it>homologues. Multiple alignment showed a premature stop codon, which prevents the encoding of the conserved cysteine residue at the <it>E2 </it>active site, and an intron that is spliced in <it>E2 </it>homologues, but not in <it>GhGDRP85</it>. The <it>GhGDRP85 </it>gene is highly expressed in different organs of cotton plants, and has high transcript levels in roots. Its promoter (uceApro2) and the 5'UTR compose a regulatory region named uceA1.7, and were isolated from cotton and studied in <it>Arabidopsis thaliana</it>. uceA1.7 shows strong expression levels, equaling or surpassing the expression levels of CaMV35S. The uceA1.7 regulatory sequence drives GUS expression 7-fold higher in flowers, 2-fold in roots and at similar levels in leaves and stems. GUS expression levels are decreased 7- to 15-fold when its 5'UTR is absent in uceApro2.</p> <p>Conclusions</p> <p>uceA1.7 is a strong constitutive regulatory sequence composed of a promoter (uceApro2) and its 5'UTR that will be useful in genetic transformation of dicots, having high potential to drive high levels of transgene expression in crops, particularly for traits desirable in flower and root tissues.</p
Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis
Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays
EFEITO DO TRATAMENTO COM NIMESULIDA SOBRE A INFLAMAÇÃO GRANULOMATOSA EM CAMUNDONGOS
O granuloma induzido por corpo estranho é um modelo experimental de infamação subcrônica que permite a avaliação do efeito de fármacos sobre o extravasamento vascular (edema) e sobre o infltrado celular. A Nimesulida é um anti-infamatório não esteroidal (AINE) que age por vários mecanismos e é largamente utilizada por sua boa tolerabilidade e comodidade de administração. O objetivo deste trabalho foi verifcar o efeito da Nimesulida sobre o modelo de granuloma induzido por corpo estranho. Camundongos Swiss machos (n=6/grupo) receberam implantes subcutâneos de algodão previamente pesados e esterilizados. O grupo Nimesulida foi tratado com o fármaco na dose de 5mg/kg a cada 12 horas, por via oral, por seis dias. Os animais do grupo Controle receberam igual volume de água estéril e apirogênica por via oral, nos mesmos intervalos e pelo mesmo período. Um dia após o período de tratamento, os animais foram sacrifcados, os implantes de algodão retirados e obtido o peso úmido. Os implantes foram pressionados contra lâminas, posteriormente coradas e utilizadas para a contagem diferencial de células. Após dessecagem por 48 horas em estufa, foi obtido o peso seco. Houve diminuição signifcativa do peso úmido do implante e signifcativa alteração do padrão do infltrado celular do granuloma, com diminuição de neutróflos e aumento de macrófagos nos animais tratados com Nimesulida, que apresentaram um padrão tissular de infamação mais tardia se comparados aos animais do grupo Controle. Assim, o tratamento com Nimesulida modula a resposta infamatória no modelo de granuloma por corpo estranho, podendo nesses casos ser utilizada como controle positivo.Descritores: Inflamação; Granuloma; Nimesulida.Abstract: The cotton pellet granuloma is a subchronic infammation experimental model that allows to assessing the effect of drugs on the vascular leakage (edema) and on the cell infltrate. Nimesulide is a NSAID that acts by several mechanisms and is widely used for its good tolerability and convenience of administration. This experiment aim was to verify the effect of Nimesulide on cotton pellet granuloma. Swiss male mice separated in two groups (n=6/group) received subcutaneous implants of cotton previously weighed and sterilized and began orally treatment with Nimesulide 5mg/kg at interval of 12 hours for six days. Control group animals received an equal volume of sterile and pyrogen-free water orally at the same interval and for the same period. One day after the treatment, the animals were sacrifced and had the implants removed. The wet weight was obtained and the implants pressed against glass slides, then stained and used for differential cell count. There was a signifcant decrease at the wet weigh and a signifcant change in the pattern of granuloma cellular infltrate, with replacement of neutrophils by macrophages in animal treated with Nimesulide. This group showed a more chronic tissue quality compared to the Control group. Then, treatment with Nimesulide is able to modulate the infammation in foreign body granuloma model and can be used as positive control.Descriptors: Infammation; Granuloma; Nimesulide
Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions
Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress
Variability of systemic and oro-dental phenotype in two families with non-lethal Raine syndrome with FAM20C mutations
Background: Raine syndrome (RS) is a rare autosomal recessive bone dysplasia typified by osteosclerosis and dysmorphic facies due to FAM20C mutations. Initially reported as lethal in infancy, survival is possible into adulthood. We describe the molecular analysis and clinical phenotypes of five individuals from two consanguineous Brazilian families with attenuated Raine Syndrome with previously unreported features. Methods: The medical and dental clinical records were reviewed. Extracted deciduous and permanent teeth as well as oral soft tissues were analysed. Whole exome sequencing was undertaken and FAM20C cDNA sequenced in family 1. Results: Family 1 included 3 siblings with hypoplastic Amelogenesis Imperfecta (AI) (inherited abnormal dental enamel formation). Mild facial dysmorphism was noted in the absence of other obvious skeletal or growth abnormalities. A mild hypophosphataemia and soft tissue ectopic mineralization were present. A homozygous FAM20C donor splice site mutation (c.784 + 5 g > c) was identified which led to abnormal cDNA sequence. Family 2 included 2 siblings with hypoplastic AI and tooth dentine abnormalities as part of a more obvious syndrome with facial dysmorphism. There was hypophosphataemia, soft tissue ectopic mineralization, but no osteosclerosis. A homozygous missense mutation in FAM20C (c.1487C > T; p.P496L) was identified. Conclusions: The clinical phenotype of non-lethal Raine Syndrome is more variable, including between affected siblings, than previously described and an adverse impact on bone growth and health may not be a prominent feature. By contrast, a profound failure of dental enamel formation leading to a distinctive hypoplastic AI in all teeth should alert clinicians to the possibility of FAM20C mutations
Measurement of the cosmic ray spectrum above eV using inclined events detected with the Pierre Auger Observatory
A measurement of the cosmic-ray spectrum for energies exceeding
eV is presented, which is based on the analysis of showers
with zenith angles greater than detected with the Pierre Auger
Observatory between 1 January 2004 and 31 December 2013. The measured spectrum
confirms a flux suppression at the highest energies. Above
eV, the "ankle", the flux can be described by a power law with
index followed by
a smooth suppression region. For the energy () at which the
spectral flux has fallen to one-half of its extrapolated value in the absence
of suppression, we find
eV.Comment: Replaced with published version. Added journal reference and DO
Energy Estimation of Cosmic Rays with the Engineering Radio Array of the Pierre Auger Observatory
The Auger Engineering Radio Array (AERA) is part of the Pierre Auger
Observatory and is used to detect the radio emission of cosmic-ray air showers.
These observations are compared to the data of the surface detector stations of
the Observatory, which provide well-calibrated information on the cosmic-ray
energies and arrival directions. The response of the radio stations in the 30
to 80 MHz regime has been thoroughly calibrated to enable the reconstruction of
the incoming electric field. For the latter, the energy deposit per area is
determined from the radio pulses at each observer position and is interpolated
using a two-dimensional function that takes into account signal asymmetries due
to interference between the geomagnetic and charge-excess emission components.
The spatial integral over the signal distribution gives a direct measurement of
the energy transferred from the primary cosmic ray into radio emission in the
AERA frequency range. We measure 15.8 MeV of radiation energy for a 1 EeV air
shower arriving perpendicularly to the geomagnetic field. This radiation energy
-- corrected for geometrical effects -- is used as a cosmic-ray energy
estimator. Performing an absolute energy calibration against the
surface-detector information, we observe that this radio-energy estimator
scales quadratically with the cosmic-ray energy as expected for coherent
emission. We find an energy resolution of the radio reconstruction of 22% for
the data set and 17% for a high-quality subset containing only events with at
least five radio stations with signal.Comment: Replaced with published version. Added journal reference and DO
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