Accelerated decarboxylation of 1,3-dimethylorotic acid in ionic liquid

The solvent effect of ionic liquids on the decarboxylation of 1,3-dimethylorotic acid and its analogue in ionic was investigated. The rate acceleration observed was proposed to be a result of the stabilization of the zwitterionic intermediates by the charged groups available in these special solvents.     

Response of human rhabdomyosarcoma cell lines to retinoic acid: relationship with induction of differentiation and retinoic acid sensitivity

The ability of retinoids to induce growth inhibition associated with differentiation of diverse cell types makes them potent anti-cancer agents. We examined the effect of retinoic acid (RA) in cell lines derived from rhabdomyosarcoma (RMS), a malignant soft-tissue tumor committed to the myogenic lineage, but arrested prior to terminal differentiation. We showed that several RMS derived cell lines, including RD human rhabdomyosarcoma cells, are resistant to the growth-inhibitory and differentiation effects of RA. We established that this RA-resistance correlates with reduced expression and activity of RA-receptors in RD cells. We stably expressed either RARalpha, RARbeta, RARgamma, or RXRalpha expression vector into RD cells and found that only RARbeta or RARgamma induced a significant RA growth arrest without promoting differentiation indicating that changes in the amounts of RARs and RXRs are not sufficient to determine the RA myogenic response of rhabdomyosarcoma cells. Activation of RD cell differentiation by ectopic MRF4 expression enhanced RA-receptor activity and led to RA induction of differentiation. These studies demonstrate that RA-resistance of RD cells is linked to their lack of differentiation and suggest that the differentiation-promoting activity of RA requires factors other than RAR-RXR heterodimers.     

A robust characterization of retinoic acid response elements based on a comparison of sites in three species

The availability of high-throughput genomic sequencing has allowed us to construct a more robust characterization of retinoic acid response elements than was possible in the past. We located human, mouse, and rat homologs for each of 51 well-documented, conserved retinoic acid response elements. Mathematical and statistical analyses of these 153 sites, 78 of which are new, shows that 92% of response elements have direct-repeat symmetry, but that only 76% exhibit canonical spacing attributes. While the familiar ‘(a/g)g(g/t)tca’ hexamer motif is upheld, the more relaxed sequence, ‘(a/g)g(g/t)(g/t)(g/c)a’, represents a 10% consensus. Sites are as likely to be on the coding strand as on the non-coding strand, and 86% of them are in upstream locations. From a statistical point of view, DR1 elements are fundamentally different from DR2 and DR5 elements, but this is only evident in the 5′ hexamer. While there is considerable variation in core positions, and while no nucleotide can be considered forbidden at any position, variation among species at a fixed locus appears surprisingly constrained once a functional site has been attained.     

Peroxidase catalysed oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone

Horseradish peroxidase catalysed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to methoxy-p-benzoquinone. Peroxidase also catalysed the oxidation of vanillyl alcohol to vanillin and vanillic acid; however, neither vanillyl alcohol nor vanillin appeared to give rise to methoxyhydroquinone directly. Correspondingly, peroxidase catalysed the oxidative decarboxylation of syringic acid to 2,6-dimethoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to 2,6-dimethoxy-p-benzoquinone.     

Differential cellular distribution of retinoic acid during staurosporine potentiation of retinoic acid-induced granulocytic differentiation in human leukemia HL-60 cells

Pretreatment of cells with staurosporine, a protein kinase C (PKC) inhibitor, was found to potentiate the granulocytic differentiation induced by a brief (2 h) retinoic acid treatment. By cell cycle analysis, staurosporine was found to have little effect on the cell cycle. Retinoic acid was distributed equally in the nuclei (40%) and in the plasma membrane (40%) of staurosporine-pretreated cells while less than 20% of retinoic acid was found in the membrane of control non-staurosporine-pretreated cells during the retinoic acid-induced differentiation. These results indicate that the enhancing effect of staurosporine may be somehow associated with the localization of retinoic acid in the plasma membrane of the cell. -1This work is dedicated to Prof. Harris Busch (Baylor College of Medicine, Tex., USA) for his 33 wonderful years at Baylor and 50 years in medicine.     

Immunolocalization of retinoic acid biosynthesis systems in selected sites in rat

Vitamin A deficiency leads to focal metaplasia of numerous epithelial tissues with altered differentiation from columnar (in general) to stratified squamous cells. This process can be reversed with vitamin A repletion. Previously, we described a system of retinoic acid (RA) synthesis in the cycling rat uterus consisting of cellular retinol binding protein (Crbp), epithelial retinol dehydrogenase (eRoldh), retinal dehydrogenase 2 (Aldh1a2), and cellular retinoic acid binding protein type II (Crabp2). Western blot analysis, RT-PCR, and immunohistochemistry were performed to test whether this retinoic acid synthesis system was also present in other vitamin A sensitive tissues. We found that combinations of Crbp, eRoldh, Aldh1a2 or Aldh1a3, and Crabp2 were present in all vitamin A sensitive tissues examined. In the ureter, while eRoldh was present, another short chain alcohol dehydrogenase reductase (possibly Roldh 1, 2, or 3) was in higher concentration in the transitional epithelia. In several tissues, Crbp, Aldh1a2, and/or Aldh1a3 localized to mesenchyme and/or epithelial cells, while eRoldh and Crabp2 were expressed only in epithelial cells. This suggests that mesenchymal-epithelial interactions may be as important in the adult as they are during development and that local synthesis of RA is important in maintenance of these tissues.     

Induction of human aquaporin-1 gene by retinoic acid in human erythroleukemia HEL cells

Retinoids have been implicated in the control of cell proliferation, differentiation, and developmental processes. We report here that aquaporin-1 (AQP1) is specifically induced by retinoic acid (RA) in human erythroleukemia HEL cells. Both all-trans-RA (ATRA) and 9-cis-RA (9CRA) strongly induced the AQP1 mRNA and protein in a dose-dependent manner. AQP1 protein was mainly expressed in plasma membrane in cells induced by RAs. To identify the RA response element (RARE) in the human AQP1 promoter, the 5(')-flanking region of AQP1 promoter was isolated and transient transfection experiment in HEL cells was performed. Deletion analysis of the AQP1 promoter revealed that one putative DR5-like RARE with five spaces was located in the region from -2218 to -2202; AGGGCAgggacAGGTGA. Electrophoretic mobility shift assay (EMSA) experiment demonstrated that two slowly migrated complexes (C1 and C2) capable of binding the RARE sequence were present in nuclear extracts prepared from cells and the complex C1 was strongly increased in nuclear extracts by RA stimulation. The complexes C1 and C2 were significantly abolished by an excess unlabeled probe. These results indicate that RAs strongly stimulate the human AQP1 gene expression through the RARE and define a novel role in the regulation of erythropoiesis.     

Expression and function of a retinoic acid receptor in budding ascidians

 Retinoic acid is thought to induce transdifferentiation of multipotent epithelial stem cells in the developing buds of the ascidian Polyandrocarpa misakiensis. We isolated a cDNA clone from this species, named PmRAR, encoding a retinoic acid receptor (RAR) homologue. PmRAR clusters with other RARs on phylogenetic trees constructed by three different methods. Within the cluster, PmRAR is on a separate branch from all the subtypes of RARs, suggesting that RAR subtypes arose in the ancestral vertebrates after divergence of vertebrates and urochordates. The embryos of another ascidian species Ciona intestinalis were co-electroporated with a mixture of a PmRAR expression vector and a lacZ reporter plasmid containing vertebrate-type retinoic acid response elements. The expression of lacZ depended on the presence of both retinoic acid and PmRAR, suggesting that PmRAR is a functional receptor. PmRAR mRNA is expressed in the epidermis and mesenchyme cells of the Polyandrocarpa developing bud. The mRNA is not detectable in the mesenchyme cells in the adult body wall, but its expression can be induced by retinoic acid in vitro. These results suggest that the PmRAR is a mediator of retinoic acid signalling in transdifferentiation during asexual reproduction of protochordates. Received: 6 April 1998 / Accepted: 27 July 1998     

Apoptotic and anti-proliferative effects of all-trans retinoic acid. Adenine nucleotide translocase sensitizes HeLa cells to all-trans retinoic acid

We examined the apoptotic and anti-proliferative effects of all-trans retinoic acid (atRA) in HeLa cells. Our results demonstrated that HeLa cells were more sensitive to the anti-proliferative effects of atRA than to its apoptotic effects. Furthermore, we demonstrated that caspase inhibition attenuates cell death but does not alter the atRA-dependent reduction in cell proliferation, which suggests that atRA-induced apoptosis is independent of the arrest in cell proliferation. To check whether ANT proteins mediated these atRA effects, we transiently transfected cells with expression vectors encoding for individual ANT (adenine nucleotide translocase 1-3). Our results revealed that ANT1 and ANT3 over-expressing HeLa cells increased their atRA sensitivity. Thus, our results not only demonstrate the different functional activities of ANT isoforms, but also contribute to a better understanding of the properties of atRA as an anti-tumoral agent used in cancer therapy.     

Excessive retinoic acid inhibit mouse embryonic palate mesenchymal cell growth through involvement of Smad signaling

All-trans retinoic acid (atRA), the oxidative metabolite of retinoic acid (RA), is essential for palatogenesis. Overdose RA is capable of inducing cleft palate in mice and humans. Normal embryonic palatal mesenchymal (EPM) cell growth is crucial for shelf growth. Smad signaling is involved in many biological processes. However, it is not much clear if atRA could affect Smad signaling during EPM cells growth. In this study, the timed pregnant mice with maternal administration of 100?mg/kg body weight of RA by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by hematoxylin and eosin (H&E) staining. At the same time, a primary mouse EPM (MEPM) cell culture model was also established. MEPM cells were treated with atRA (0.1, 0.5, 1, 5 and 10?μM) for 24, 48 and 72?h. The results indicated that the sizes of the shelves were smaller than those in control. AtRA inhibited MEPM cell growth with both increasing concentration and increasing incubation time, especially at 72?h in vitro. Moreover, atRA significantly increased the mRNA and protein expression levels of Smad7 (P?<?.05), but the mRNA and protein expression levels of PCNA were reduced (P?<?.05). We also found atRA inhibited phosphorylation of Smad2 compared with untreated group (P?<?.05). However, the protein and mRNA levels of Smad2 did not change both in atRA-treated and untreated group (P?>?.05). We demonstrated that RA induced inhibition of MEPM cell growth that could cause cleft palate partly by down-regulation of Smad pathway.     

Regulation of selenoprotein mRNA expression by hormones and retinoic acid in bovine mammary cells

Selenium is essential for maintaining many body functions through the actions of selenoproteins. To find factors regulating selenoprotein biosynthesis in the bovine mammary cell line MAC-T, the effects of supplementation with selenite and also with retinoic acid, insulin, hydrocortisone and prolactin on the mRNA expression of a number of selenoproteins were investigated. It was found that MAC-T cells express glutathione peroxidase (GPx) 1 and 4, thioredoxin reductase 1 and selenoprotein P, but not GPx 3, which is interesting considering that GPx 3 is one of the only few selenoproteins detected in milk so far. Addition of selenite to the cell culture resulted in a large increase in GPx 1 expression and an increase in selenoprotein P expression, which is similar to the findings made in other systems investigated. Increased mRNA levels of GPx 1 were also observed in cells treated with insulin and hydrocortisone or with retinoic acid. The expression of thioredoxin reductase 1 was increased in cells treated with retinoic acid, whereas that of selenoprotein P was decreased in cells exposed to insulin. The results indicate that several hormones, selenium, and retinoic acid regulate the biosynthesis of various selenoproteins differently in the bovine mammary cell. The possible implications of the findings for processes related to milk formation and mammary carcinogenesis will need additional investigation. Further study of the detailed mechanisms involved is also necessary.     

Atomic-resolution STM structure of DNA and localization of the retinoic acid binding site

Single-molecule imaging by scanning tunnelling microscopy (STM) yields the atomic-resolution (0.6A) structure of individual B-type DNA molecules. The strong correlation between these STM structures and those predicted from the known base sequence indicates that sequencing of single DNA molecules using STM may be feasible. There is excellent agreement between the STM and X-ray structures, but subtle differences exist due to radial distortions. We show that the interactions of other molecules with DNA, their binding configurations, and the structure of these complexes can be studied at the single-molecule level. The anti-cancer drug retinoic acid (RA) binds selectively to the minor groove of DNA with up to 6 RA molecules per DNA turn and with the plane of the RA molecule approximately parallel to the DNA symmetry axis. Similar studies for other drug molecules will be valuable in the a priori evaluation of the effectiveness of anti-cancer drugs.     

In vitro differentiation of mouse embryonic stem cells after activation by retinoic acid

Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of blastocysts. ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm, and ectoderm) of the organism, including the germline. In recent reports mouse ES cells have been successfully applied in the treatment of spinal cord injury, hereditary myelin disorder of the central nervous system, and diabetes mellitus. In this study, we investigated the induction of mouse ES cell differentiation, using culture of embryoid bodies (EBs) into the diverse tissues. EBs were formed by culturing ES cells (129/SV strain) in DMEM supplemented with 10% FBS, in the absence of feeder cells and leukemia inhibitory factor (LF). EBs were induced to differentiate by treatment with retinoic acid (RA). In control medium (non-RA medium) beating muscles, blood vessels, hemocytes, and cartilages were frequently observed in EBs. Moreover, when EBs were cultured in medium including RA (5 x 10(-8) M, and 5 x 10(-9) M), differentiation of the optic vesicle, lens, retina, and neural groove was observed. In this study we demonstrated that an efficient system for inducing the differentiation of ES cells using EBs.     

Dhrs3a regulates retinoic acid biosynthesis through a feedback inhibition mechanism

Retinoic acid (RA) is an important developmental signaling molecule responsible for the patterning of multiple vertebrate tissues. RA is also a potent teratogen, causing multi-organ birth defects in humans. Endogenous RA levels must therefore be tightly controlled in the developing embryo. We used a microarray approach to identify genes that function as negative feedback regulators of retinoic acid signaling. We screened for genes expressed in early somite-stage embryos that respond oppositely to treatment with RA versus RA antagonists and validated them by RNA in situ hybridization. Focusing on genes known to be involved in RA metabolism, we determined that dhrs3a, which encodes a member of the short-chain dehydrogenase/reductase protein family, is both RA dependent and strongly RA inducible. Dhrs3a is known to catalyze the reduction of the RA precursor all-trans retinaldehyde to vitamin A; however, a developmental function has not been demonstrated. Using morpholino knockdown and mRNA over-expression, we demonstrate that Dhrs3a is required to limit RA levels in the embryo, primarily within the central nervous system. Dhrs3a is thus an RA-induced feedback inhibitor of RA biosynthesis. We conclude that retinaldehyde availability is an important level at which RA biosynthesis is regulated in vertebrate embryos.