Assessment of activated gut-homing CD8+ T cells in blood by flow cytometry during a 3-day gluten challenge.

Accurate celiac disease (CD) diagnosis must be performed in individuals following a gluten containing diet. Diagnostic procedures for individuals already on a gluten-free diet (GFD) avoiding long gluten reintroductions are still challenging. To deal with this issue, we developed an accurate but simple method that requires only a 3-day gluten challenge and circumvents the main limitations of previously suggested proposals such as requirement of specific peptides and unusual specialized lab facilities or high cost. In an attempt to standardize this methodology to be used in daily clinical practice, we describe here an optimized protocol for assessing activated gut-homing CD8+ T cells in blood combined with a short gluten challenge. Details about the amount and type of gluten antigen and the starting material are included, as well as the strategy to easily characterize and identify the cells of interest using flow cytometry. This methodology constitutes a diagnostic tool for CD diagnosis of high specificity and sensitivity for seropositive disease (>95%) as an alternative to long-term gluten challenge and open new possibilities to test the response to gluten in research and clinical trials.

Crop photoperiodism model 2.0 for the flowering time of sorghum and rice that includes daily changes in sunrise and sunset times and temperature acclimation.

BACKGROUND AND AIMS: Daylength determines flowering dates. However, questions remain regarding flowering dates in the natural environment, such as the synchronous flowering of plants sown simultaneously at highly contrasting latitudes. The daily change in sunrise and sunset times is the cue for the flowering of trees and for the synchronization of moulting in birds at the equator. Sunrise and sunset also synchronize the cell circadian clock, which is involved in the regulation of flowering. The goal of this study was to update the photoperiodism model with knowledge acquired since its conception. METHODS: A large dataset was gathered, including four 2-year series of monthly sowings of 28 sorghum varieties in Mali and two 1-year series of monthly sowings of eight rice varieties in the Philippines to compare with previously published monthly sowings in Japan and Malaysia, and data from sorghum breeders in France, Nicaragua and Colombia. An additive linear model of the duration in days to panicle initiation (PI) and flowering time using daylength and daily changes in sunrise and sunset times was implemented. KEY RESULTS: Simultaneous with the phyllochron, the duration to PI of field crops acclimated to the mean temperature at seedling emergence within the usual range of mean cropping temperatures. A unique additive linear model combining daylength and daily changes in sunrise and sunset hours was accurately fitted for any type of response in the duration to PI to the sowing date without any temperature input. Once calibrated on a complete and an incomplete monthly sowing series at two tropical latitudes, the model accurately predicted the duration to PI of the concerned varieties from the equatorial to the temperate zone. CONCLUSIONS: Including the daily changes in sunrise and sunset times in the updated photoperiodism model largely improved its accuracy at the latitude of each experiment. More research is needed to ascertain its multi-latitudinal accuracy, especially at latitudes close to the equator.

Pediatric Klüver-Bucy Syndrome: Report of Two Cases and Review of the Literature.

Klüver-Bucy syndrome (KBS) is a rare behavioral phenotype described in monkeys and humans that appears most often after bilateral temporal damage. The main features of KBS are compulsion to examine objects orally, increased sexual activity, placidity, hypermetamorphosis, visual agnosia, and amnesia. Cases in children are scarce, and the most frequently reported etiology is herpes encephalitis. Hyperorality (90%), hypersexuality (82%), and epilepsy (70%) were the most common features of the 51 cases reported in the literature to date. Carbamazepine, selective serotonin reuptake inhibitors (SSRIs), and neuroleptics have been used for symptomatic treatment with variable control. Corticosteroids or immunosupressive agents, such as rituximab, can be an option to use in some cases, according to etiology suspicion. Cognitive and behavioral disturbances after KBS are often severe, but improvement can occur over a long time and residual disabilities vary from major to fairly mild.We report two new encephalitis-associated pediatric patients and review all of the pediatric KBS cases in the literature to better describe the clinical features of this rare neurobehavioral condition.

Exposure to particulate matter air pollution and risk of multiple sclerosis in two large cohorts of US nurses.

BACKGROUND: Air pollution is thought to raise the risk of neurological disease by promoting neuroinflammation, oxidative stress, glial activation and cerebrovascular damage. Multiple Sclerosis is a common auto-immune disorder, primarily affecting young women. We conducted, to a large prospective study of particulate matter (PM) exposure and multiple sclerosis (MS) risk in two prospective cohorts of women: the Nurses Health Study (NHS) and the Nurses Health Study II (NHS II). METHODS: Cumulative average exposure to different size fractions of PM up to the onset of MS was estimated using spatio-temporal models. We used multivariable Cox proportional hazards models to estimate the hazard ratios (HR) and 95% confidence intervals (CI) of MS associated with each size fraction of PM independently. Participants were followed from 1998 through 2004 in NHS and from 1988 through 2007 for NHS II. We conducted additional sensitivity analyses stratified by smoking, region of the US, and age, as well as analyses restricted to women who did not move during the study. Analyses were adjusted for age, ancestry, smoking, body mass index at age 18, region, tract level population density, latitude at age 15, and UV index. RESULTS: We did not observe significant associations between air pollution and MS risk in our cohorts. Among women in the NHS II, the HRs comparing the top vs. bottom quintiles of PM was 1.11 (95% Confidence Intervals (CI): 0.74, 1.66), 1.04 (95% CI: 0.73, 1.50) and 1.09 (95% CI: 0.73, 1.62) for PM10 (≤10µm in diameter), PM2.5 (≤2.5µm in diameter), and PM2.5-10 (2.5 to 10µm in diameter) respectively, and tests for linear trends were not statistically significant. No association between exposure to PM and risk of MS was observed in the NHS. CONCLUSIONS: In this study, exposure to PM air pollution was not related to MS risk.

Different Gene Expression Signatures in Children and Adults with Celiac Disease.

Celiac disease (CD) is developed after gluten ingestion in genetically susceptible individuals. It can appear at any time in life, but some differences are commonly observed between individuals with onset early in life or in adulthood. We aimed to investigate the molecular basis underlying those differences. We collected 19 duodenal biopsies of children and adults with CD and compared the expression of 38 selected genes between each other and with the observed in 13 non-CD controls matched by age. A Bayesian methodology was used to analyze the differences of gene expression between groups. We found seven genes with a similarly altered expression in children and adults with CD when compared to controls (C2orf74, CCR6, FASLG, JAK2, IL23A, TAGAP and UBE2L3). Differences were observed in 13 genes: six genes being altered only in adults (IL1RL1, CD28, STAT3, TMEM187, VAMP3 and ZFP36L1) and two only in children (TNFSF18 and ICOSLG); and four genes showing a significantly higher alteration in adults (CCR4, IL6, IL18RAP and PLEK) and one in children (C1orf106). This is the first extensive study comparing gene expression in children and adults with CD. Differences in the expression level of several genes were found between groups, being notorious the higher alteration observed in adults. Further research is needed to evaluate the possible genetic influence underlying these changes and the specific functional consequences of the reported differences.

The association between metabolic syndrome and prostate cancer: Effect on its aggressiveness and progression.

OBJECTIVES: To evaluate the impact of metabolic syndrome and its individual components on prostate biopsy findings, the radical prostatectomy specimen and on biochemical recurrence. MATERIAL AND METHODS: An observational study was conducted of 1319 men who underwent prostate biopsy between January 2007 and December 2011. The impact on the biopsy findings, the radical prostatectomy specimen and biochemical recurrence was evaluated using logistic regression and Cox regression. RESULTS: Of the 1319 patients, 275 (21%) had metabolic syndrome, and 517 prostate cancers were diagnosed. A greater percentage of metabolic syndrome was found among patients with prostate cancer than among patients without prostate cancer (25% vs. 18%; P=.002). Poorer results were found in the radical prostatectomy specimens (Gleason score ≥ 7, P<.001; stage ≥ T2c, P<.001; positive surgical margins, P<.001), and there was a greater percentage of biochemical recurrence in patients with metabolic syndrome than in those without metabolic syndrome (24% vs. 13%; P=.003). Metabolic syndrome behaved as an independent predictive factor of finding a Gleason score ≥ 7 for the specimen, as well as for finding a specimen stage ≥ T2c. Metabolic syndrome was also able to independently predict a greater rate of biochemical recurrence (OR: 3.6, P<.001; OR: 3.2, P=.03; HR: 1.7; respectively). CONCLUSIONS: Metabolic syndrome is associated with poorer findings in the radical prostatectomy specimens and is an independent prognostic factor of biochemical recurrence.

[Development of a high content protein beverage from Chilean mesquite, lupine and quinoa for the diet of pre-schoolers]. / Desarrollo de una bebida de alto contenido proteico a partir de algarrobo, lupino y quinoa para la dieta de preescolares.

This research was aimed at developing a high content protein beverage from the mixture of liquid extracts of a pseudocereal, quinoa (Chenopodium quinoa Willd) and two legumes: mesquite (Prosopis chilensis (Mol.) Stunz) and lupine (Lupinus albus L.), native from the Andean highlands of the Chilean northern macro-zone, flavored with raspberry pulp, to help in the feeding of children between 2 and 5 years of lower socioeconomic status with nutritional deficiencies. The formulation was defined by linear programming, its composition was determined by proximate analysis and physical, microbiological and sensory acceptance tests were performed. After 90 days of storage time, the beverage got a protein content of 1.36%, being tryptophan the limiting amino acid; for its part, the chromaticity coordinates of CIEL*a*b* color space showed no statistical significant differences (p < 0.05) maintaining the "dark pink" tonality, the viscosity and the sensory evaluation were acceptable for drinking.

[Product development on the basis of cereal and leguminous flours to coeliac disease in children between 6-24 months; I: formulation and acceptability]. / Desarrollo de producto sobre la base de harinas de cereales y leguminosa para niños celíacos entre 6 y 24 meses; I: formulación y aceptabilidad.

The revaluation of the Andean cultivations, quinua (Chenopodium quinua Willd) and lupin (Lupinus albus L.), to be used in nutritional mixtures, with traditional cereals like corn (Zea mays L.) and rice (Oryza sativa L.), originate mixtures without gluten which constitute a good alternative for the nutrition of children under 24 months that suffer from celiac disease, since they improve the quality of the protein, by essential amino acids compensation, and also impacts in the product's diversification strategy. In the present work, the percentage composition of each flour in the mixture was determined by means of Linear Programming by means of the Solver form from the Excel spreadsheet. Prolamines were determined in the quinua and lupin flours by the ELISA test and the HPLC technique was used in both products obtained called "sweet mix" and "dessert mix", to define the quantity of amino acids with the purpose of providing around the 15% of the proteins required in the day. The flour mixtures selected as optimum, sweet mix, suitable for the preparation of sweet pancakes, as well as for the dessert mix, that by addition of water or milk produce a semi solid dessert, were evaluated after three months of storage, being acceptable their microbiological, bromatological and sensorial requirements, corroborating the results with the good acceptance of the products, prepared from the formulated mixtures, by the children of two Day Care centers of the City of Antofagasta-Chile.

Polymorphisms of caffeine metabolism and estrogen receptor genes and risk of Parkinson's disease in men and women.

Caffeine intake has been associated with a decreased risk of Parkinson's disease (PD) in men but the effect in women is less clear, and appears to be modified by use of post-menopausal estrogens. In a nested case-control study within the Nurses Health Study (NHS) and the Health Professionals Follow-up Study (HPFS), we examined associations between single nucleotide polymorphisms (SNPs) of caffeine metabolizing genes (CYP1A2 and NAT2) and estrogen receptors (ESR1 and ESR2), their interaction with caffeine intake and hormone replacement therapy (PMH) use (collected prospectively) and risk of PD. We matched 159 female cases to 724 controls and 139 male cases to 561 controls on birth year, source of DNA (blood or buccal smear), age and sex. The CYP1A2 rs762551 polymorphism (lower enzyme inducibility) was marginally associated with an increased risk of PD (RR, for increasing number of minor alleles=1.34; 95% CI 1.02, 1.78 in women, but not in men. None of the NAT2 (classified as slow vs. fast acetylator), ESR1 or ESR2 polymorphisms were significantly associated with an altered risk of PD. Marginally significant interactions were observed between caffeine intake and the ESR1 polymorphism rs3798577 (p=0.07) and ESR2 polymorphism rs1255998 (p=0.07). The observed increased risk of PD among female but not male carriers of the rs762551 polymorphism of CYP1A2 and the interactions of caffeine with ESR1 rs3798577 and ESR2 rs1255998 may provide clues to explain the relationship between gender, caffeine intake, estrogen status and risk of PD and need to be replicated.

[Adalimumab induction and maintenance therapy for Crohn's disease. An open-label study]. / Tratamiento de induccion y mantenimiento con adalimumab en la enfermedad de Crohn: un estudio abierto.

BACKGROUND: adalimumab has been shown in placebo-controlled clinical trials and uncontrolled studies to be effective in luminal and perianal fistulizing CD. OBJECTIVE: to evaluate the efficacy and safety of adalimumab for induction and maintenance therapy in CD. METHODS: twenty-two patients with CD treated with adalimumab (16 for luminal disease and 6 for active perianal fistulizing disease) were included. Twenty-one patients had previously received IFX. All patients received induction therapy with 160 mg s.c. at week 0, and 80 mg s.c. at week 2. Responders received maintenance therapy with 40 mg s.c. every 14 days. Response was assessed at 4 weeks after the initial dose, and classified as remission, partial response, or non-response. RESULTS: after induction, 25% of patients with luminal disease had a complete remission, and 56.3% had a partial response. Clinical response was maintained in 71.6% of patients at 1 year, in 53.7% at 18 months, and in 35.8% at 48 months. No differences in response were observed between patients with hypersensitivity reactions or loss of response to IFX.All patients with perianal fistulizing disease (n = 6) had been previously treated with IFX. After induction 16.7% entered remission, and 66.7% had a partial response. All patients maintained remission or response over time, with a median follow-up of 15 months. CONCLUSIONS: adalimumab is an effective and safe treatment for the induction and maintenance of response in luminal and perianal fistulizing CD. These results confirm that the findings obtained in controlled clinical trials are reproducible in clinical practice.

IGF-I and vasoactive intestinal peptide (VIP) regulate cAMP-response element-binding protein (CREB)-dependent transcription via the mitogen-activated protein kinase (MAPK) pathway in pituitary cells: requirement of Rap1.

In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10(-7) M) or VIP (10(-7) M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.

IGF-I inhibits apoptosis through the activation of the phosphatidylinositol 3-kinase/Akt pathway in pituitary cells.

In previous studies we demonstrated that IGF-I induces proliferation of pituitary lactotrophs. In addition to its mitotrophic actions, IGF-I is known to prevent apoptosis induced by diverse stimuli in several cell types. In this study, we investigated the action of IGF-I on pituitary cell survival and the intracellular signaling transduction pathway implicated in this effect. Treatment of cultured male rat pituitary cells with IGF-I (10(-7) M) for 24 h prevented pituitary cell death induced by serum deprivation. The protective effect of IGF-I was blocked by phosphoinositide 3-kinase (PI3-kinase) inhibitor, LY294002, but was unaffected by PD98059, which inhibits MAP/ERK kinase (MEK1). IGF-I activation of PI3-kinase induced the phosphorylation and activation of the serine/threonine kinase Akt. Moreover, IGF-I increased the phosphorylation of the pro-apoptotic factor Bad and the levels of the anti-apoptotic protein Bcl-2 through the PI3-kinase pathway in primary pituitary cells.

Clinical and genetic linkage analysis of a large Venezuelan kindred with Usher syndrome.

OBJECTIVE: To undertake a comprehensive investigation into the very high incidence of congenital deafness on the Macano peninsula of Margarita Island, Venezuela. METHODS: Numerous visits were made to the isolated island community over a 4-year-period. During these visits, it became apparent that a significant number of individuals complained of problems with hearing and vision. Socioeconomic assessments, family pedigrees and clinical histories were recorded on standard questionnaires. All individuals underwent thorough otolaryngologic and ophthalmologic examinations. Twenty milliliters of peripheral venous blood was obtained from each participant. A genome-wide linkage analysis study was performed. Polymorphic microsatellite markers were amplified by polymerase chain reaction and separated on polyacrylamide gels. An ABI 377XL sequencer was used to separate fragments and LOD scores were calculated by using published software. RESULTS: Twenty-four families were identified, comprising 329 individuals, age range 1-80 years, including 184 children. All families were categorized in the lower two (least affluent) socioeconomic categories. A high incidence of consanguinity was detected. Fifteen individuals (11 adults, 4 children) had profound congenital sensorineural hearing loss, vestibular areflexia and retinitis pigmentosa. A maximum LOD score of 6.76 (Linkage >3.0), between markers D11s4186 and D11s911, confirmed linkage to chromosome 11q13.5. The gene myosin VIIA (MYO7A) was confirmed in the interval. Clinical and genetic findings are consistent with a diagnosis of Usher syndrome 1B for those with hearing and vision problems. CONCLUSIONS: We report 15 Usher syndrome 1B individuals from a newly detected Latin American socio-demographic origin, with a very high prevalence of 76 per 100,000 population.

[Medical care of patients with heart failure: clinical characteristics, determinants of prognosis and follow-up in primary care]. / La atención médica de los pacientes con insuficiencia cardíaca: características clínicas, determinantes del pronóstico y seguimiento en la atención primaria.

OBJECTIVE: To assess the process of care and prognosis of patients with heart failure (HF) attended in a tertiary hospital and follow up at the primary care level. DESIGN: Prospective study of 18 months of follow up.Setting. Tertiary hospital and primary care centers of the reference area. Participants. Patients admitted to a tertiary hospital from the first of july until de 31 of december of 1998. OUTCOME MEASUREMENTS: Pharmacological data and morbimortality at discharge and at the end of the follow-up, functional capacity of survivors. RESULTS: 265 patients were included, with a mean age of 75 years, 57% were females, 73.8% had HF as first diagnosis, 6.1% had MI, and 20% were attended for other medical reasons. The most frequent cause of HF was HTA. Drugs more prescribed at the discharge and follow up were diuretics and ACE inhibitors. Hospital mortality was 6.4% and mortality at the end of the follow-up was 46% (in 77% of those for cardiac reasons). After being discharged 38.5% of the patients were readmitted to the hospital with the diagnosis of HF, 72% were visited by the family physician, 43% at the outpatient clinic and 33% by the cardiologist; 60% of the patients who survived were in I-II NYHA functional class, 76% walked regularly, and 25% did recreational activities and physical exercise. CONCLUSIONS: Patients attended at the hospital with HF are an old population, have frequently associated other chronic diseases, and have a very bad prognosis. These patients spend an important amount of health resources. Drug prescription at the follow up is suboptimum. Patients who survived have an acceptable functional capacity.

Involvement of VIP on BDNF-induced somatostatin gene expression in cultured fetal rat cerebral cortical cells.

Previous studies have shown that BDNF promotes expression of SS. In earlier studies we demonstrated the stimulatory effect of locally produced VIP upon SS secretion. These facts led us to explore the peptidergic action of BDNF on VIP, and to determine if BDNF-induced SS might be mediated by the induction of VIP. Cultured fetal rat cerebrocortical cells were incubated with BDNF (50 ng/ml) and/or VIP (10(-11) M) for 2 and 5 days. In other experiments IgGs from BDNF or VIP antisera were also added. BDNF increased VIP and SS gene expression and peptide production. After 2 days of incubation with both BDNF and VIP the induction of SS mRNA was similar to that obtained with BDNF alone. However when the treatment was extended to 5 days the increase in SS mRNA was higher than that obtained with BDNF alone. This finding suggests the possibility that both factors acted synergistically. To define the potential role of VIP in the response of SS gene expression to BDNF, endogenous VIP was blocked with IgGs from VIP antiserum. Under these experimental conditions BDNF-induced SS decreased. Our study provides the first evidence that BDNF up-regulates VIP gene expression and concentration of the peptide. The involvement of VIP on BDNF-induced SS gene expression is also demonstrated.

Mechanisms of reduced body growth in the pubertal feminized male rat: unbalanced estrogen and androgen action on the somatotropic axis.

It is well known that the sex difference in body growth at puberty is modulated by a complex interplay between sex steroids and somatotropic axis; however, the exact role played by sex steroids remains a matter of controversy. The aim of this study was to assess the mechanisms by which sex steroids regulate body growth during pubertal development. Flutamide, a non-steroid-blocking androgen receptor, was subcutaneously administered to 30-d-old male Wistar rats for 4 wk. The blockade of the androgen receptor led to a marked elevation in serum testosterone and an increment in serum estradiol. Flutamide administration decreased body weight gain, serum IGF-I levels, hepatic IGF-I mRNA, and GH receptor mRNA content. There were no significant changes in serum GH concentration, pituitary GH reserve, and pituitary GH mRNA content. Flutamide lowered hypothalamic somatostatin mRNA content and augmented hypothalamic immunoreactive somatostatin stores, but did not alter hypothalamic immunoreactive GH-releasing factor stores. Our findings indicate that during pubertal development of the male rat, the imbalance between androgen and estrogen actions determines an abnormal somatic growth, which is at least partly exerted through the peripheral or hepatic modification of the somatotropic axis that occurs under the high or exclusive action of estrogens. Potential implication of coincident sex-specific regulated mode of pulsatile GH secretion cannot be excluded from this random serum GH sample study.

Potassium depolarization-induced cAMP stimulates somatostatin mRNA levels in cultured diencephalic neurons.

In the nervous system, signals transmitted across synapses are known to regulate gene expression in the postsynaptic cells. This process often involves membrane depolarization and subsequent elevation of intracellular Ca(2+). We have previously demonstrated in fetal cerebrocortical cells, that somatostatin (SS) mRNA levels can be induced by depolarizing agents such as high potassium concentrations and veratridine (VTD), and that these effects are calcium dependent. SS expression is regulated by cAMP, and in the cerebral cortex adenylate cyclase activity is regulated through fluctuations in intracellular Ca(2+) concentrations. The present experiments were undertaken to determine the mechanism by which calcium upregulates the levels of SS mRNA. Cerebrocortical cells from 17-day-old fetuses were exposed to the different agents for 24 h and the levels of SS mRNA were measured by Northern blot. Incubation of cells with the calcium channel antagonist nifedipine (Nf), the calcium chelating agent EGTA, calcium free KRB and the calcium calmodulin inhibitors trifluoroperazine (TFP) and the napthelene sulfonamide, W7, resulted in the inhibition of K(+)-induced SS mRNA levels. K(+)-evoked depolarization increased the intracellular concentration of cAMP and this effect was antagonized by verapamil (VPM). Forskolin (Fk) provoked a higher increment in cAMP concentration than potassium, although the induction of SS mRNA was more evident following K(+) depolarization indicating a lack of correlation between levels of cAMP and induction of SS mRNA. The role of K(+)-induced cAMP on the increment of SS mRNA that occurred upon membrane depolarization was further explored with the inhibitor of protein kinase A (PKA), Rp cAMP whose presence significantly reduced depolarization-induced SS mRNA levels. This study confirms that Ca(2+) influx is required for K(+)depolarization-induced stimulation of cAMP whereby the increment of SS mRNA is partly produced.

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.

[Impact of biomedical publications on the incorporation of new therapeutic attitudes in cardiovascular pathology]. / Impacto de las publicaciones biomédicas en la incorporación de nuevas actitudes terapéuticas en patología cardiovascular.

OBJECTIVE: To evaluate the application degree of results from three clinical trials on cardiovascular pathology in clinical practice: SOLVD trial (in patients with congestive heart failure), SAVE trial (in patients with acute myocardial infarction) and SPINAF trial (in patients with chronic atrial fibrillation). DESIGN: Retrospective cross-sectional study performed in the first six months in 1990 and 1992 (SOLVD trial) and in the first six months in 1991 and 1993 (SAVE trial) admitted to the Consorci Hospitalari del Parc Taulí, and a cross-sectional study in a single randomized sample of all patients with the discharge diagnosis of atrial fibrillation at Hospital Vall d'Hebron during 1994. An absolute increase of 23% and 19% in the prescription of ACEI agents was observed for patients with heart failure and myocardial infarction, respectively. Forty-eight percent of patients with atrial fibrillation received antithrombotic therapy, which included aspirin and acenocoumarine for 51% and 49% of cases, respectively.