Construction of miRNA–mRNA networks for the identification of lung cancer biomarkers in liquid biopsies

Lung cancer (LC) is the most common cause of cancer death worldwide mostly due to the low survival rate: 75% of cases are identified in advanced stages. In this study, the list of useful biomarkers to make an early diagnosis using liquid biopsies was expanded. A total of 30 samples of LC were analyzed to define potential miRNA biomarkers in liquid biopsies for LC. The biomarkers have been identified in interaction networks miRNA–mRNA. The potential biomarkers have been then validated in large cohorts. A total of 15 candidate miRNAs, that regulate the repression of 30 mRNAs, have been identified as a specific functional interaction network for squamous carcinoma, while the specific functional interaction network of adenocarcinoma consists of four candidate miRNAs that seem to handle the repression of five mRNA. Inspection of expression levels in larger cohorts validates the usefulness of the 11 candidates as biomarkers in liquid biopsies. The 11 candidate miRNAs found could be utilized to form diagnostic predictive biomarkers for LC in liquid biopsies (AU)

Construction of miRNA-mRNA networks for the identification of lung cancer biomarkers in liquid biopsies.

Lung cancer (LC) is the most common cause of cancer death worldwide mostly due to the low survival rate: 75% of cases are identified in advanced stages. In this study, the list of useful biomarkers to make an early diagnosis using liquid biopsies was expanded. A total of 30 samples of LC were analyzed to define potential miRNA biomarkers in liquid biopsies for LC. The biomarkers have been identified in interaction networks miRNA-mRNA. The potential biomarkers have been then validated in large cohorts. A total of 15 candidate miRNAs, that regulate the repression of 30 mRNAs, have been identified as a specific functional interaction network for squamous carcinoma, while the specific functional interaction network of adenocarcinoma consists of four candidate miRNAs that seem to handle the repression of five mRNA. Inspection of expression levels in larger cohorts validates the usefulness of the 11 candidates as biomarkers in liquid biopsies. The 11 candidate miRNAs found could be utilized to form diagnostic predictive biomarkers for LC in liquid biopsies.

EVOO Promotes a Less Atherogenic Profile Than Sunflower Oil in Smooth Muscle Cells Through the Extracellular Vesicles Secreted by Endothelial Cells.

Background: Little is known about the effect of extra virgin olive (EVOO) and sunflower oil (SO) on the composition of extracellular vesicles (EVs) secreted by endothelial cells and the effects of these EVs on smooth muscle cells (SMCs). These cells play an important role in the development of atherosclerosis. Methods: We evaluated the effects of endothelial cells-derived EVs incubated with triglyceride-rich lipoproteins obtained after a high-fat meal with EVOO (EVOO-EVs) and SO (SO-EVs), on the transcriptomic profile of SMCs. Results: We found 41 upregulated and 19 downregulated differentially expressed (DE)-miRNAs in EVOO-EVs. Afterwards, SMCs were incubated with EVOO-EVs and SO-EVs. SMCs incubated with SO-EVs showed a greater number of DE-mRNA involved in pathways related to cancer, focal adhesion, regulation of actin cytoskeleton, and MAPK, toll-like receptor, chemokine and Wnt signaling pathways than in SMCs incubated with EVOO-EVs. These DE-mRNAs were involved in biological processes related to the response to endogenous stimulus, cell motility, regulation of intracellular signal transduction and cell population proliferation. Conclusion: EVOO and SO can differently modify the miRNA composition of HUVEC-derived EVs. These EVs can regulate the SMCs transcriptomic profile, with SO-EVs promoting a profile more closely linked to the development of atherosclerosis than EVOO-EVs.

An Integrative Approach of an In Vitro Measurement of the Digestibility of Triacylglycerols of Human Milk.

Several studies have been published regarding the effect of different factors on the digestion of milk lipids, considering their natural structural arrangement as milk fat globules and the efficiency of the digestive enzymes in the lipolysis of such complex structures. During digestion, the lipolytic products are dispersed in vesicles and micelles, which are the source for absorption of digested lipids. Therefore, it is necessary to consider the isolation of the micellar phase from the digesta to appropriately determine the amounts and classes of lipids which are bioaccessible. This study presents an integrative approach that included an isolation procedure to separate the micellar fraction from undigested and non-micellar parts, and the distribution of digested milk lipids in micelles determined directly through chromatographic techniques. Four groups of five full term mothers donated colostrum or mature milk. Two sets of samples were analyzed directly (raw), and two sets were pasteurized and then analyzed. Our data revealed that the profile of digested milk lipids is different depending on the lactation period and processing stage, while the carbon atom number distribution of the digested triacylglycerols in the micellar fraction provides a substantial information regarding the acylglycerols species that are less available for absorption.

NGS of microRNAs Involved in Cardioprotection Induced by Sevoflurane Compared to Propofol in Myocardial Revascularization Surgery: The ACDHUVV-16 Clinical Trial.

BACKGROUND: Numerous studies have demonstrated that halogenated agents elicit myocardial conditioning effects when administered perioperatively in cardiac surgery. Recent evidence has been published on the benefits of maintaining exposure to halogenated agents during the early postoperative period. The enzymatic mechanisms by which this beneficial effect is exerted were explained recently. OBJECTIVES: Our study was performed to investigate whether this phenomenon is mediated by either the activation or suppression of miRNAs targeted by halogenated anesthetics. METHODS: A double-blind, two-stage trial was conducted. The results of the first stage of the trial are presented in this paper. The sample was composed of patients undergoing off-pump myocardial revascularization surgery. Patients were randomized to receive either sevoflurane [S] or propofol [P] during the intraoperative and early postoperative period (during the first six hours after the intervention). Hemodynamics (heart rate, blood pressure, central venous pressure, cardiac index, systolic volume index, LVEF) and myocardial enzymes (troponin I) were monitored at six hour intervals during the first 48 hours. In the first stage of the trial, blood was drawn for gene sequencing from eight patients (four per group) at baseline and at 24 h. In the second stage of the study, a qPCR analysis was performed of the miRNAs identified as significant by gene sequencing. Levels of cardioprotective enzymes (serine/threonine protein kinase (Akt), tumor necrosis factor alpha (TNFα), extracellular regulated protein kinase (ERK 1/2), and caspase 3) were measured to assess their role in myocardial conditioning pathways. The purpose was to identify the miRNAs that play a major role in myocardial conditioning induced by halogenated agents. Concentrations of cardioprotective enzymes were higher in patients who received sevoflurane than the patients who were administered propofol. RESULTS: NGS differences were observed between baseline and 24-h values in the two study groups. In group P, miRNA 197-3p was overexpressed, whereas miRNAs 4443 and 1294, 708-3p were underexpressed. In group S, miRNAs 615-3p, 4466, 29, 937-3p, 636, 197-3P, 184, 4685, 296-3p, 147b, 3199, 6815, 1294 and 3176 were underexpressed; whereas 708-3p was overexpressed. qPCR showed significant variations in miRNAs 197-3p, 4443, 708-3p and 1294 in the P group, and in miRNAs 937-3p, 636, 197- 3p, 296-3p and 708-3p in the S group. CONCLUSION: In the P Group, changes in the expression of some miRNAs were associated with lower concentrations of the enzymes involved in myocardial pre- and postconditioning. In contrast, in Group S, variations in miRNAs were associated with the activation of mediators of anesthetic-induced pre- and post-conditioning, a reduction in cell apoptosis, and a decrease in caspase and TnBF alpha concentrations. Changes in these miRNAs were associated with better prognosis in patients with ischemic heart disease. The main limitation of this study will be overcome in the second stage of the trial, where the specific role of each miRNA will be determined.

miRNA/Target Gene Profile of Endothelial Cells Treated with Human Triglyceride-Rich Lipoproteins Obtained after a High-Fat Meal with Extra-Virgin Olive Oil or Sunflower Oil.

SCOPE: The effects of triglyceride-rich lipoproteins (TRLs) on the miRNA expression of endothelial cells, which are very involved in atherosclerosis, according to the type of diet are not known. METHODS AND RESULTS: The differences between the effects of TRLs isolated from blood of subjects after a high-fat meal with extra-virgin olive oil (EVOO) and sunflower oil (SO) on the microRNA-Seq profile related to atherosclerosis in human umbilical vein endothelial cells are analyzed. 28 upregulated microRNAs with EVOO-derived TRLs, which can regulate 22 genes related to atherosclerosis, are found. 21 upregulated microRNAs with SO-derived TRLs, which can regulate 20 genes related to atherosclerosis, are found. These microRNAs are mainly involved in angiogenesis, with a predominance of an anti-angiogenic effect with EVOO-derived TRLs. Other microRNAs upregulated with SO-derived TRLs are involved in cardiovascular diseases. Pathways for the target genes obtained from the upregulated microRNA with EVOO-derived TRLs are involved in lipid metabolism and inflammatory and defense response, while those with SO-derived TRLs are involved in lipid metabolic process. CONCLUSION: EVOO-derived TRLs seem to produce a more atheroprotective profile than SO-derived TRLs. This study provides alternative mechanisms on the protective role of EVOO against the atherogenic process through microRNA regulation in endothelial cells.

Effect of gestational age (preterm or full term) on lipid composition of the milk fat globule and its membrane in human colostrum.

Human colostrum is the first milk secreted by the mother after birth and constitutes the ideal food for the newborn, because its chemical composition, rich in immunoglobulins, antimicrobial peptides, growth factors, bioactive lipids, and other important molecules, is perfectly adapted to the metabolic, digestive, and immunological immaturity of the newborn. An incomplete gestational period can affect the maturity of the mammary gland and its ability to secrete milk with the proper composition for the newborn's condition. Previous studies indicate that the mammary gland modulates the profiles of bioactive lipids present in the different phases of lactation from colostrum to mature milk. Given the key role played by the polar lipids (PL) (phospho- and sphingolipids) of the milk fat globule membrane (MFGM) in the immune system and cognitive development of the newborn, it is crucial to analyze whether the content and distribution of the PL are affected by gestation period. Therefore, this study aimed to determine the milk fat globule (MFG) and MFGM lipid compositions of human colostrum samples from 20 healthy preterm and full-term mothers. Lipid characterization using chromatographic techniques (gas chromatograph mass spectrometry and HPLC-evaporative light-scattering detection) revealed differences related to length of gestation in the profiles of lipid classes and fatty acid and triacylglyceride contents of colostrum. This comparative analysis leads to noteworthy outcomes about the changing roles of the PL, considering the preterm or full-term condition. We found a lack of correlation of some PL (such as phosphatidylcholine, phosphatidylinositol, and phosphatidylserine) with the delivery term; these could be denoted as structural category lipids. However, sphingomyelin and phosphatidyl-ethanolamine exhibited trends to decrease in full-term colostrum, indicating that in the final stage of pregnancy specific accretion of some PL occurs, which should be denoted as a nutritional redistribution.

In Vitro Digestion of Human Milk: Influence of the Lactation Stage on the Micellar Carotenoids Content.

Human milk is a complex fluid with nutritive and non-nutritive functions specifically structured to cover the needs of the newborn. The present study started with the study of carotenoid composition during progress of lactation (colostrum, collected at 3-5 d postpartum; mature milk, collected at 30 d postpartum) with samples donated from full-term lactating mothers (women with no chronic diseases, nonsmokers on a regular diet without supplements, n = 30). Subsequently, we applied an in vitro protocol to determine the micellarization efficiency of the carotenoids, which were separated by HPLC and quantified by the external standard method. That in vitro protocol is tailored for the biochemistry of the digestive tract of a newborn. To the best of our knowledge, the present study is the first report of carotenoids micellar contents, obtained in vitro. This study reveals, from the in vitro perspective, that colostrum and mature milk produce significant micellar contents of carotenoids despite lipids in milk are within highly complex structures. Indeed, the lactation period develops some influence on the micellarization efficiency, influence that might be attributed to the dynamics of the milk fat globule membrane (MFGM) during the progress of lactation.

Carotenoid Content in Human Colostrum is Associated to Preterm/Full-Term Birth Condition.

Factors such as lactation stage and premature and small-for-gestational conditions could lead to great inter-individual variability in the carotenoid content of human milk. The aim was to analyze the carotenoid content in colostrum and mature milk of preterm (PT) and full-term (FT) mothers to establish whether they are significantly different and, if so, the stage of lactation when the differences are established. Samples of blood, colostrum, and mature milk were collected from Spanish donating mothers who gave birth to PT or FT infants. Carotenoids from serum and milk samples were analyzed by HPLC-atmospheric pressure chemical ionization (APCI)-MS. Quantitatively, colostrum from PT mothers presented lower total carotenoid content when compared to that from FT mothers. The only exception was lutein, where levels were not different. The transition from colostrum to mature milk makes observed differences in the carotenoid content disappear, since there were no variances between PT and FT groups for both individual and total carotenoid content. The premature birth condition affects the quantitative carotenoid composition of the colostrum but has no effect on the lutein content. This fact could be related to the significant role of this xanthophyll in the development of infant retina and feasibly to cognitive function.

Xanthophyll esters are found in human colostrum.

SCOPE: Carotenoids in human milk are associated with other lipid counterparts in several metabolic processes. One interesting association that has not been demonstrated to date is the presence of xanthophyll esters. Colostrum and mature milk samples were analyzed to determine the occurrence of xanthophyll esters and identify the compounds. Thus, the association of the amounts of these compounds with lactation and whether they are significant contributors to the carotenoid profile of human milk was assessed. METHODS AND RESULTS: Pre-term and term delivering mothers were included in the study to donate colostrum at 3-5 days postpartum and mature milk at 15 days postpartum. Carotenoids extracts were subjected to a clean-up procedure to remove the triacylglycerol fraction and then analyzed by HPLC-MSn . Identification of xanthophyll esters was achieved by considering their chromatographic behaviour, UV-visible characteristics and MSn features. CONCLUSION: Xanthophyll esters are significant contributors to the carotenoid profile in the colostrum, while mature milk does not contain these compounds. Therefore, fatty acid acylation to xanthophylls is activated during the accumulation of carotenoids in the human mammary gland. The sharp decline in the amount of xanthophyll esters in mature milk indicates that the lipophilic components are those recently incorporated in the mammary epithelium.

Adaptación de las recomendaciones internacionales en estabilización y reanimación neonatal 2015 / Changes in the international recommendations on neonatal stabilisation and resuscitation (2015)

Las recomendaciones internacionales del International Liaison Committee on Resuscitation (ILCOR), mediante una revisión exhaustiva de la evidencia disponible en el desarrollo de las medidas de soporte a la transición y de reanimación del recién nacido tras su nacimiento, aportan una guía universal a partir de la cual cada grupo o comité local puede adaptarla a su realidad e idiosincrasia, y elaborar sus propias guías o recomendaciones. El objetivo de esta revisión es analizar los principales cambios, abordar las controversias generadas desde 2010, contrastarlas con las de otras organizaciones nacionales e internacionales como son la European Resuscitation Council (ERC), American Heart Association (AHA) o la Australian-New Zealand Committee on Resuscitation (ANZCOR). De esta forma, el Grupo de Reanimación Neonatal de la Sociedad Española de Neonatología (GRN-SENeo) consensúa respuestas claras sobre muchas de las preguntas que ofrecen diferentes opciones de actuación, y genera las próximas recomendaciones de nuestro país para el soporte a la transición o la reanimación del recién nacido tras su nacimiento, con seguridad y eficacia

The International Liaison Committee on Resuscitation (ILCOR) recommendations provide a universal guide of measures to support the transition and resuscitation of newborn after their birth. This guide is expected to be adapted by local groups or committees on resuscitation, according to their own circumstances. The objective of this review is to analyse the main changes, to discuss several of the controversies that have appeared since 2010, and contrasting with other national and international organisations, such as European Resuscitation Council (ERC), American Heart Association (AHA), or the Australian-New Zealand Committee on Resuscitation (ANZCOR). Thus, the Neonatal Resuscitation Group of the Spanish Society of Neonatology (GRN-SENeo) aims to give clear answers to many of the questions when different options are available, generating the forthcoming recommendations of our country to support the transition and/or resuscitation of a newborn after birth, safely and effectively

[Changes in the international recommendations on neonatal stabilisation and resuscitation (2015)]. / Adaptación de las recomendaciones internacionales en estabilización y reanimación neonatal 2015.

The International Liaison Committee on Resuscitation (ILCOR) recommendations provide a universal guide of measures to support the transition and resuscitation of newborn after their birth. This guide is expected to be adapted by local groups or committees on resuscitation, according to their own circumstances. The objective of this review is to analyse the main changes, to discuss several of the controversies that have appeared since 2010, and contrasting with other national and international organisations, such as European Resuscitation Council (ERC), American Heart Association (AHA), or the Australian-New Zealand Committee on Resuscitation (ANZCOR). Thus, the Neonatal Resuscitation Group of the Spanish Society of Neonatology (GRN-SENeo) aims to give clear answers to many of the questions when different options are available, generating the forthcoming recommendations of our country to support the transition and/or resuscitation of a newborn after birth, safely and effectively.

Establishing gene models from the Pinus pinaster genome using gene capture and BAC sequencing.

BACKGROUND: In the era of DNA throughput sequencing, assembling and understanding gymnosperm mega-genomes remains a challenge. Although drafts of three conifer genomes have recently been published, this number is too low to understand the full complexity of conifer genomes. Using techniques focused on specific genes, gene models can be established that can aid in the assembly of gene-rich regions, and this information can be used to compare genomes and understand functional evolution. RESULTS: In this study, gene capture technology combined with BAC isolation and sequencing was used as an experimental approach to establish de novo gene structures without a reference genome. Probes were designed for 866 maritime pine transcripts to sequence genes captured from genomic DNA. The gene models were constructed using GeneAssembler, a new bioinformatic pipeline, which reconstructed over 82% of the gene structures, and a high proportion (85%) of the captured gene models contained sequences from the promoter regulatory region. In a parallel experiment, the P. pinaster BAC library was screened to isolate clones containing genes whose cDNA sequence were already available. BAC clones containing the asparagine synthetase, sucrose synthase and xyloglucan endotransglycosylase gene sequences were isolated and used in this study. The gene models derived from the gene capture approach were compared with the genomic sequences derived from the BAC clones. This combined approach is a particularly efficient way to capture the genomic structures of gene families with a small number of members. CONCLUSIONS: The experimental approach used in this study is a valuable combined technique to study genomic gene structures in species for which a reference genome is unavailable. It can be used to establish exon/intron boundaries in unknown gene structures, to reconstruct incomplete genes and to obtain promoter sequences that can be used for transcriptional studies. A bioinformatics algorithm (GeneAssembler) is also provided as a Ruby gem for this class of analyses.

Transcriptome analysis in maritime pine using laser capture microdissection and 454 pyrosequencing.

Maritime pine (Pinus pinaster Aiton) is one of the most advanced conifer models for genomics research. Conifer genomes are extremely large and major advances have recently been made in the characterization of transcriptomes. The combination of laser capture microdissection (LCM) and next-generation sequencing is a powerful tool with which to resolve the entire transcriptome of specific cell types and tissues. In the current work, we have developed a protocol for transcriptomic analyses of conifer tissue types using LCM and 454 pyrosequencing. Tissue sections were isolated using non-fixed flash-frozen samples processed by LCM. Complementary DNA synthesis and amplification from tiny amounts of total RNA from LCM samples was performed using an adapted protocol for C: onifer R: NA A: mplification (CRA+). The cDNA amplification yield and cDNA quality provided by CRA+ were adequate for 454 pyrosequencing. Furthermore, read length and quality results of the 454 runs were near the optimal parameters considered by Roche for transcriptome sequencing. Using the CRA+ protocol, non-specific amplifications were prevented, problems derived from poly(A:T) tails in the 454 sequencing technology were reduced, and read length and read number considerably enhanced. This technical approach will facilitate global gene expression analysis in individual tissues of conifers and may also be applied to other plant species.

De novo assembly of maritime pine transcriptome: implications for forest breeding and biotechnology.

Maritime pine (Pinus pinasterAit.) is a widely distributed conifer species in Southwestern Europe and one of the most advanced models for conifer research. In the current work, comprehensive characterization of the maritime pine transcriptome was performed using a combination of two different next-generation sequencing platforms, 454 and Illumina. De novo assembly of the transcriptome provided a catalogue of 26 020 unique transcripts in maritime pine trees and a collection of 9641 full-length cDNAs. Quality of the transcriptome assembly was validated by RT-PCR amplification of selected transcripts for structural and regulatory genes. Transcription factors and enzyme-encoding transcripts were annotated. Furthermore, the available sequencing data permitted the identification of polymorphisms and the establishment of robust single nucleotide polymorphism (SNP) and simple-sequence repeat (SSR) databases for genotyping applications and integration of translational genomics in maritime pine breeding programmes. All our data are freely available at SustainpineDB, the P. pinaster expressional database. Results reported here on the maritime pine transcriptome represent a valuable resource for future basic and applied studies on this ecological and economically important pine species.

Ablación de extrasistolia ventricular del tracto de salida ventricular derecho con sistema de cartografía sin contacto / Ablation of premature ventricular contractions originating in the right ventricular outflow tract using non-contact mapping

La extrasistolia ventricular originada en el tracto de salida de ventrículo derecho puede tener una respuesta pobre a fármacos y ser compleja de ablacionar mediante un sistema convencional con guía fluoroscópica debido a la dificultad en la inducibilidad. Describimos la ablación de extrasistolia ventricular de difícil inducibilidad originada en el tracto de salida de ventrículo derecho, mediante un sistema de cartografía sin contacto. Se realizó ablación de cinco focos extrasistólicos originados en tracto de salida de ventrículo derecho en una serie prospectiva de 4 pacientes. Los pacientes presentaban pobre calidad de vida y falta de respuesta a antiarrítmicos. Se realizó una media de tres aplicaciones de radiofrecuencia por foco, con un tiempo medio de aplicación de 113±15 s. Se obtuvo un 100% de éxito agudo y no hubo complicaciones. En un seguimiento medio de 30±16 meses, los pacientes se mantienen asintomáticos sin tratamiento farmacológico. El sistema de cartografía sin contacto permite una alta eficacia en la eliminación de extrasistolias ventriculares aisladas de difícil inducibilidad (AU)

Premature ventricular contractions originating in the right ventricular outflow tract may respond poorly to pharmacological treatment, and ablation using conventional fluoroscopically-guided systems may be complicated by the difficulty in inducing arrhythmias. We describe the use of a non-contact mapping system to ablate difficult-to-induce premature ventricular contractions originating in the right ventricular outflow tract. Five premature ventricular contractions sites in the right ventricular outflow tract were ablated in a prospective series of 4 patients. Patients had a poor quality of life and had not responded to antiarrhythmic drugs. A mean of 3 radiofrequency pulses per site was applied and mean application time was 113±15s. We achieved a 100% acute success rate and there were no complications. Patients were asymptomatic without drug therapy after a mean of 30±16 months of follow-up. The noncontact mapping system is highly effective in eliminating difficult to induce, isolated premature ventricular contractions (AU)

[Ablation of premature ventricular contractions originating in the right ventricular outflow tract using non-contact mapping]. / Ablación de extrasistolia ventricular del tracto de salida ventricular derecho con sistema de cartografía sin contacto.

Premature ventricular contractions originating in the right ventricular outflow tract may respond poorly to pharmacological treatment, and ablation using conventional fluoroscopically-guided systems may be complicated by the difficulty in inducing arrhythmias. We describe the use of a non-contact mapping system to ablate difficult-to-induce premature ventricular contractions originating in the right ventricular outflow tract. Five premature ventricular contractions sites in the right ventricular outflow tract were ablated in a prospective series of 4 patients. Patients had a poor quality of life and had not responded to antiarrhythmic drugs. A mean of 3 radiofrequency pulses per site was applied and mean application time was 113±15s. We achieved a 100% acute success rate and there were no complications. Patients were asymptomatic without drug therapy after a mean of 30±16 months of follow-up. The noncontact mapping system is highly effective in eliminating difficult to induce, isolated premature ventricular contractions.

Functional interactions between a glutamine synthetase promoter and MYB proteins.

In Scots pine (Pinus sylvestris), ammonium assimilation is catalysed by glutamine synthetase (GS) [EC 6.3.1.2], which is encoded by two genes, PsGS1a and PsGS1b. PsGS1b is expressed in the vascular tissue throughout the plant body, where it is believed to play a role in recycling ammonium released by various facets of metabolism. The mechanisms that may underpin the transcriptional regulation of PsGS1b were explored. The PsGS1b promoter contains a region that is enriched in previously characterized cis-acting elements, known as AC elements. Pine nuclear proteins bound these AC element-rich regions in a tissue-specific manner. As previous experiments had shown that R2R3-MYB transcription factors could interact with AC elements, the capacity of the AC elements in the PsGS1b promoter to interact with MYB proteins was examined. Two MYB proteins from loblolly pine (Pinus taeda), PtMYB1 and PtMYB4, bound to the PsGS1b promoter were able to activate transcription from this promoter in yeast, arabidopsis and pine cells. Immunolocalization experiments revealed that the two MYB proteins were most abundant in cells previously shown to accumulate PsGS1b transcripts. Immunoprecipitation analysis and supershift electrophoretic mobility shift assays implicated these same two proteins in the formation of complexes between pine nuclear extracts and the PsGS1b promoter. Given that these MYB proteins were previously shown to have the capacity to activate gene expression related to lignin biosynthesis, we hypothesize that they may function to co-regulate lignification, a process that places significant demands on nitrogen recycling, and GS, the major enzyme involved in the nitrogen recycling pathway.

Molecular analysis of the 5'-upstream region of a gibberellin-inducible cytosolic glutamine synthetase gene (GS1b) expressed in pine vascular tissue.

The promoter region of the cytosolic glutamine synthetase GS1b gene was isolated from the conifer Pinus sylvestris L. (Scots pine). The 1,171-bp stretch of sequence lying immediately upstream of the transcriptional start site was sufficient to drive the expression of a reporter gene in a manner consistent with the expression pattern of the native GS1b gene. Computer analysis of putative cis elements in this promoter region revealed the presence of an AT box, an AC motif similar to those found in other genes expressed in the vascular tissue, and a gibberellin (GA)-responsive element. Consistent with the latter finding, GS1b gene expression was induced by exogenously supplied gibberellic acid (GA3) in germinating pine embryos and pine seedlings. In order to examine if the putative GA-response element found in the GS1b promoter could function in the regulation of GS1b expression, a series of deletions of the upstream gene region were fused to the uidA reporter gene, and transient expression analyzed either in untreated or in GA3-treated pine (Pinus pinaster Ait.) protoplasts. Deletion analysis revealed that sequences containing the GA-responsive element, located between -1005 and -724 bp were essential for the increased promoter activity observed in response to GA3. Furthermore, electrophoretic-mobility-shift assays showed that pine nuclear proteins bind to a 22-bp sequence that contains the GA-response element, located between -768 and -747 bp relative to the transcription start site.