ABSTRACT
Previous research showed that berberine-containing Berberis species synthesise the substances 5'-methoxyhydnocarpin-D (5'-MHC-D) and pheophorbide a, which have no antimicrobial activity but inhibit the expression of multidrug resistant efflux pumps (MDRs) in Staphylococcus aureus and potentiate the action of berberine. The MDR pumps extrude synthetic and natural antimicrobials from bacterial cells. We searched for these compounds in Berberis aetnensis C. Presl. (Berberidaceae), an endemic plant of the volcano Mount Etna. This work confirms the presence of pheophorbide a and permits us to hypothesise the presence of 5'-MHC-D in leaf extracts. In fact, the activity of ciprofloxacin was improved when two chromatographic fractions isolated from leaf extracts were added. These results are indicative of the presence of MDR pump inhibitors. Moreover, crude extracts were tested on several micro-organisms and showed antimicrobial activity mainly against Gram-positive bacteria and yeasts.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Berberis , Chlorophyll/analogs & derivatives , Anti-Bacterial Agents/administration & dosage , Anti-Bacterial Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Berberine/administration & dosage , Berberine/chemistry , Berberine/isolation & purification , Berberine/pharmacology , Candida/drug effects , Chlorophyll/administration & dosage , Chlorophyll/chemistry , Chlorophyll/isolation & purification , Chlorophyll/pharmacology , Ciprofloxacin/administration & dosage , Drug Interactions , Flavonoids/administration & dosage , Flavonoids/chemistry , Flavonoids/isolation & purification , Flavonoids/pharmacology , Gram-Negative Bacteria/drug effects , Gram-Positive Bacteria/drug effects , Molecular Structure , Multidrug Resistance-Associated Proteins , Plant Extracts/pharmacology , Staphylococcus aureus/drug effectsABSTRACT
How gene activity is translated into phenotype and how it can modify morphogenetic pathways is of central importance when studying the evolution of regulatory control mechanisms. Previous studies in mouse have suggested that, despite the homeodomain-restricted homology, Drosophila orthodenticle (otd) and murine Otx1 genes share functional equivalence and that translation of Otx2 mRNA in epiblast and neuroectoderm might require a cell type-specific post-transcriptional control depending on its 5' and 3' untranslated sequences (UTRs). In order to study whether OTD is functionally equivalent to OTX2 and whether synthesis of OTD in epiblast is molecularly dependent on the post-transcriptional control of Otx2 mRNA, we generated a first mouse model (otd(2)) in which an Otx2 region including 213 bp of the 5' UTR, exons, introns and the 3' UTR was replaced by an otd cDNA and a second mutant (otd(2FL)) replacing only exons and introns of Otx2 with the otd coding sequence fused to intact 5' and 3' UTRs of Otx2. otd(2) and otd(2FL) mRNAs were properly transcribed under the Otx2 transcriptional control, but mRNA translation in epiblast and neuroectoderm occurred only in otd(2FL) mutants. Phenotypic analysis revealed that visceral endoderm (VE)-restricted translation of otd(2) mRNA was sufficient to rescue Otx2 requirement for early anterior patterning and proper gastrulation but it failed to maintain forebrain and midbrain identity. Importantly, epiblast and neuroectoderm translation of otd(2FL) mRNA rescued maintenance of anterior patterning as it did in a third mouse model replacing, as in otd(2FL), exons and introns of Otx2 with an Otx2 cDNA (Otx2(2c)). The molecular analysis has revealed that Otx2 5' and 3' UTR sequences, deleted in the otd(2) mRNA, are required for nucleo-cytoplasmic export and epiblast-restricted translation. Indeed, these molecular impairments were completely rescued in otd(2FL) and Otx2(2c) mutants. These data provide novel in vivo evidence supporting the concept that during evolution pre-existing gene functions have been recruited into new developmental pathways by modifying their regulatory control.
Subject(s)
Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/genetics , 3' Untranslated Regions , 5' Untranslated Regions , Active Transport, Cell Nucleus , Animals , Biological Evolution , Body Patterning/genetics , Brain/embryology , Brain/metabolism , Cytoplasm/metabolism , DNA, Complementary/genetics , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins , Mice , Mice, Knockout , Morphogenesis , Otx Transcription Factors , Phenotype , Protein Biosynthesis , Species SpecificityABSTRACT
Otx genes play an important role in brain development. Previous mouse models suggested that the untranslated regions (UTRs) of Otx2 mRNA may contain regulatory element(s) required for its post-transcriptional control in epiblast and neuroectoderm. In order to study this, we have perturbed the 3' UTR of Otx2 by inserting a small fragment of DNA from the lambda phage. Otx2(lambda) mutants exhibited proper gastrulation and normal patterning of the early anterior neural plate, but from 8.5 days post coitum they developed severe forebrain and midbrain abnormalities. OTX2 protein levels in Otx2(lambda) mutants were heavily reduced in the epiblast, axial mesendoderm and anterior neuroectoderm but not in the visceral endoderm. At the molecular level, we found out that the ability of the Otx2(lambda) mRNA to form efficient polyribosome complexes was impaired. Sequence analysis of the Otx2-3' UTR revealed a 140 bp long element that is present only in vertebrate Otx2 genes and conserved in identity by over 80%. Our data provide experimental evidence that murine brain development requires accurate translational control of Otx2 mRNA in epiblast and neuronal progenitor cells. This leads us to hypothesise that this control might have important evolutionary implications.
Subject(s)
3' Untranslated Regions , Gene Expression Regulation, Developmental , Homeodomain Proteins , Mesencephalon/embryology , Nerve Tissue Proteins/genetics , Prosencephalon/embryology , Trans-Activators/genetics , Animals , Biological Evolution , Body Patterning , Conserved Sequence , Ectoderm/metabolism , Female , Gastrula , Head/abnormalities , Head/embryology , Humans , Male , Mesencephalon/metabolism , Mice , Mutation , Nerve Tissue Proteins/physiology , Otx Transcription Factors , Polyribosomes/metabolism , Prosencephalon/metabolism , Sequence Alignment , Trans-Activators/physiology , Transcription, GeneticABSTRACT
Previous mouse models have indicated that Otx1 and Otx2 play an important role in brain and sense organ development and, together with the Drosophila orthodenticle (otd) gene, they share a high degree of reciprocal functional equivalence. Interestingly, mouse models replacing the same region of the Otx2 locus with Otx1, otd or lacZ genes have revealed the existence of a differential post-transcriptional control between the visceral endoderm (VE) and epiblast cells. Indeed Otx1, otd or lacZ mRNA were transcribed in both tissues but translated only in the VE. Embryos lacking OTX1 or OTD proteins in the epiblast and derived tissues, such as the neuroectoderm and axial mesendoderm (AME), fail to maintain the anterior identity and result in a headless phenotype. This finding leads us to hypothesise that, during evolution, the specification of the vertebrate-type brain may have required epiblast cells to translate Otx2 mRNA in order to establish maintenance properties. The establishment of this regulatory control might have been reflected into a remarkable reorganisation of the rostral CNS architecture and might have represented an important event in the evolution of the vertebrate head. Current data suggest that the Otx2 replaced region and in particular the 3' untranslated region (UTR), may contain regulatory element(s) necessary to translate and/or stabilise Otx2 mRNA in epiblast and its derivatives.
Subject(s)
Biological Evolution , Brain/embryology , Nerve Tissue Proteins/genetics , Trans-Activators/genetics , Transcription Factors , Vertebrates/embryology , 3' Untranslated Regions , Animals , Conserved Sequence , Drosophila , Drosophila Proteins , Gastrula/physiology , Gene Deletion , Homeodomain Proteins/genetics , Mice , Morphogenesis/genetics , Otx Transcription FactorsABSTRACT
Most of the gene candidates for the control of developmental programmes that underlie brain morphogenesis in vertebrates are the orthologues of Drosophila genes coding for signalling molecules or transcription factors. Among these, the orthodenticle group, including the Drosophila orthodenticle (otd) and the vertebrate Otx1 and Otx2 genes, is mostly involved in fundamental processes of anterior neural patterning. In mouse, Drosophila and intermediate species otd/Otx genes have shown a remarkable similarity in expression pattern suggesting that they could be part of a conserved control system operating in the brain and different from that coded by the HOX complexes controlling the hindbrain and spinal cord. In order to verify this hypothesis, a series of mouse models have been generated in which the functions of the murine Otx genes were: (i) fully inactivated, (ii) replaced with each other, and (iii) replaced with the Drosophila otd gene. The data obtained highlight a crucial role for the Otx genes in specification, regionalization and terminal differentiation of rostral central nervous system and lead to hypothesize that modification of their regulatory control may have influenced the morphogenesis and evolution of the brain.
