Nonhypnotic low-dose etomidate for rapid correction of hypercortisolaemia in cushing's syndrome

We determined the adrenostatic potential of low-dose nonhypnotic etomidate in six patients with Cushing's syndrome (ectopic Cushing's syndrome,n=2; Cushing's disease,n=3; bilateral adrenal adenoma,n=1). Etomidate was given as a continuous infusion for 32 h in a dose of 2.5 mg/h (n=5) or 0.3 mg/kg/h (n=3), respectively. Saline was given during a control period. The responsiveness to exogenous ACTH was studied during placebo and 7 and 31 h after commencing etomidate by administration of 250 µg 1–24 ACTH i.v. Etomidate (2.5 mg/h) led to a consistent decrease in serum cortisol in all patients from a mean of 39.4±13.3 to 21.1±5.7 µg/dl after 7 h (P<0.05 compared with placebo). After 24 h cortisol was reduced further to a mean steady state concentration of 12.3±5.7 µg/dl (P<0.05). At the end of the infusion period the cortisol increase in response to ACTH was reduced but not abolished. In contrast, a dose of 0.3 mg/kg/h etomidate induced unresponsiveness of serum cortisol to exogenous ACTH within 7 h. However, sedation was observed in two out of three patients at this dose, while during etomidate in a dose of 2.5 mg/h no side effects were seen. We conclude that low-dose non-hypnotic etomidate reduces serum cortisol to within the normal range in patients with Cushing's syndrome. The possibility to dissociate the adrenostatic effect of etomidate from its hypnotic action, the absence of side effects, and the i.v. route suggest that etomidate in a dose of 0.04–0.05 mg/kg/h may become the drug of choice for rapid initial control of hypercortisolism

Detection of the DCC gene product in normal and malignant colorectal tissues and its relation to a codon 201 mutation.

Protein expression of the putative tumour-suppressor gene DCC on chromosome 18q was evaluated in a panel of 16 matched colorectal cancer and normal colonic tissue samples together with DCC mRNA expression and allelic deletions (loss of heterozygosity, LOH). Determined by a polymerase chain reaction (PCR)-LOH assay, 12 of the 16 (75%) cases were informative with LOH occurring in 2 of the 12 cases. For DCC mRNA, transcripts could be detected in all analysed normal tissues (eight out of eight) by RT-PCR, whereas 6 of the 15 tumours were negative. DCC protein expression, investigated by immunohistochemistry using the monoclonal antibody 15041 A directed against the intracellular domain, was homogeneously positive in all normal tissue samples. In tumour tissues, no DCC protein was seen in 11 out of 16 samples (69%). For the DCC codon 201, we found a loss of a wild-type codon sequence caused by mutation or LOH in at least 8 out of 15 cases (53%) compared with the corresponding normal tissue. DCC protein expression was undetectable in eight of the nine tumours missing both wild-type codons. Only one of the five tumours with retained DCC protein expression had no detectable wild-type codon 201. In addition, 9 out of 15 normal tissue specimens were mutated in codon 201. In two out of three cases with homozygous wild-type codons in peripheral blood lymphocyte (PBL) DNA, mutations were already observed in the tumour adjacent normal colonic mucosa. We conclude that DCC immunostaining should be introduced in the clinicopathological routine because of its strong correlation with the known prognostic markers 18q LOH and mutation of codon 201

The CD3-Zeta Chimeric Antigen Receptor Overcomes TCR Hypo-Responsiveness of Human Terminal Late-Stage T Cells

Adoptive therapy of malignant diseases with tumor-specific cytotoxic T cells showed remarkable efficacy in recent trials. Repetitive T cell receptor (TCR) engagement of target antigen, however, inevitably ends up in hypo-responsive cells with terminally differentiated KLRG-1+ CD57+ CD7− phenotype limiting their therapeutic efficacy. We here revealed that hypo-responsiveness of CMV-specific late-stage CD8+ T cells is due to reduced TCR synapse formation compared to younger cells. Membrane anchoring of TCR components contributes to T cell hypo-responsiveness since dislocation of galectin-3 from the synapse by swainsonine restored both TCR synapse formation and T cell response. Transgenic expression of a CD3-zeta signaling chimeric antigen receptor (CAR) recovered hypo-responsive T cells to full effector functions indicating that the defect is restricted to TCR membrane components while synapse formation of the transgenic CAR was not blocked. CAR engineered late-stage T cells released cytokines and mediated redirected cytotoxicity as efficiently as younger effector T cells. Our data provide a rationale for TCR independent, CAR mediated activation in the adoptive cell therapy to avoid hypo-responsiveness of late-stage T cells upon repetitive antigen encounter

Environmentally induced changes in antioxidant phenolic compounds levels in wild plants

[EN] Different adverse environmental conditions cause oxidative stress in plants by generation of reactive oxygen species (ROS). Accordingly, a general response to abiotic stress is the activation of enzymatic and non-enzymatic antioxidant systems. Many phenolic compounds, especially flavonoids, are known antioxidants and efficient ROS scavengers in vitro, but their exact role in plant stress responses in nature is still under debate. The aim of our work is to investigate this role by correlating the degree of environmental stress with phenolic and flavonoid levels in stress-tolerant plants. Total phenolic and antioxidant flavonoid contents were determined in 19 wild species. Meteorological data and plant and soil samples were collected in three successive seasons from four Mediterranean ecosystems: salt marsh, dune, semiarid and gypsum habitats. Changes in phenolic and flavonoid levels were correlated with the environmental conditions of the plants and were found to depend on both the taxonomy and ecology of the investigated species. Despite species-specific differences, principal component analyses of the results established a positive correlation between plant phenolics and several environmental parameters, such as altitude, and those related to water stress: temperature, evapotranspiration, and soil water deficit. The correlation with salt stress was, however, very weak. The joint analysis of all the species showed the lowest phenolic and flavonoid levels in the halophytes from the salt marsh. This finding supports previous data indicating that the halophytes analysed here do not undergo oxidative stress in their natural habitat and therefore do not need to activate antioxidant systems as a defence against salinity.This work has been funded by the Spanish Ministry of Science and Innovation (Project CGL2008-00438/BOS), with contribution from the European Regional Development Fund. Thanks to Dr. Rafael Herrera for critical reading of the manuscript.Bautista, I.; Boscaiu, M.; Lidón, A.; Llinares Palacios, JV.; Lull, C.; Donat-Torres, MP.; Mayoral García-Berlanga, O.... (2016). Environmentally induced changes in antioxidant phenolic compounds levels in wild plants. Acta Physiologiae Plantarum. 38(1):1-15. https://doi.org/10.1007/s11738-015-2025-2S115381Agati G, Biricolti S, Guidi L, Ferrini F, Fini A, Tattini M (2011) The biosynthesis of flavonoids is enhanced similarly by UV radiation and root zone salinity in L. vulgare leaves. 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Sterility and Gene Expression in Hybrid Males of Xenopus laevis and X. muelleri

BACKGROUND: Reproductive isolation is a defining characteristic of populations that represent unique biological species, yet we know very little about the gene expression basis for reproductive isolation. The advent of powerful molecular biology tools provides the ability to identify genes involved in reproductive isolation and focuses attention on the molecular mechanisms that separate biological species. Herein we quantify the sterility pattern of hybrid males in African Clawed Frogs (Xenopus) and apply microarray analysis of the expression pattern found in testes to identify genes that are misexpressed in hybrid males relative to their two parental species (Xenopus laevis and X. muelleri). METHODOLOGY/PRINCIPAL FINDINGS: Phenotypic characteristics of spermatogenesis in sterile male hybrids (X. laevis x X. muelleri) were examined using a novel sperm assay that allowed quantification of live, dead, and undifferentiated sperm cells, the number of motile vs. immotile sperm, and sperm morphology. Hybrids exhibited a dramatically lower abundance of mature sperm relative to the parental species. Hybrid spermatozoa were larger in size and accompanied by numerous undifferentiated sperm cells. Microarray analysis of gene expression in testes was combined with a correction for sequence divergence derived from genomic hybridizations to identify candidate genes involved in the sterility phenotype. Analysis of the transcriptome revealed a striking asymmetric pattern of misexpression. There were only about 140 genes misexpressed in hybrids compared to X. laevis but nearly 4,000 genes misexpressed in hybrids compared to X. muelleri. CONCLUSIONS/SIGNIFICANCE: Our results provide an important correlation between phenotypic characteristics of sperm and gene expression in sterile hybrid males. The broad pattern of gene misexpression suggests intriguing mechanisms creating the dominance pattern of the X. laevis genome in hybrids. These findings significantly contribute to growing evidence for allelic dominance in hybrids and have implications for the mechanism of species differentiation at the transcriptome level

A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein.

In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control