Identification and characterization of the retinoic acid response elements in the human RIG1 gene promoter

The expression of retinoic acid-induced gene 1 (RIG1), a class II tumor suppressor gene, is induced in cells treated with retinoids. RIG1 has been shown to express ubiquitously and the increased expression of this gene appears to suppress cell proliferation. Recent studies also demonstrated that this gene may play an important role in cell differentiation and the progression of cancer. In spite of the remarkable regulatory role of this protein, the molecular mechanism of RIG1 expression induced by retinoids remains to be clarified. The present study was designed to study the molecular mechanism underlying the all-trans retinoic acid (atRA)-mediated induction of RIG1 gene expression. Polymerase chain reaction was used to generate a total of 10 luciferase constructs that contain various fragments of the RIG1 5'-genomic region. These constructs were then transfected into human gastric cancer SC-M1 and breast cancer T47D cells for transactivation analysis. atRA exhibited a significant induction in luciferase activity only through the -4910/-5509 fragment of the 5'-genomic region of RIG1 gene relative to the translation initiation site. Further analysis of this promoter fragment indicated that the primary atRA response region is located in between -5048 and -5403 of the RIG1 gene. Within this region, a direct repeat sequence with five nucleotide spacing, 5'-TGACCTctattTGCCCT-3' (DR5, -5243/-5259), and an inverted repeat sequence with six nucleotide spacing, 5'-AGGCCAtggtaaTGGCCT-3' (IR6, -5323/-5340), were identified. Deletion and mutation of the DR5, but not the IR6 element, abolished the atRA-mediated activity. Electrophoretic mobility shift assays with nuclear extract from atRA-treated cells indicated the binding of retinoic acid receptor (RAR) and retinoid X receptor (RXR) heterodimers specifically to this response element. In addition to the functional DR5, the region contains many other potential sequence elements that are required to maximize the atRA-mediated induction. Taken together, we have identified and characterized the functional atRA response element that is responsible for the atRA-mediated induction of RIG1 gene.     

T-box binding protein type two (TBX2) is an immediate early gene target in retinoic-acid-treated B16 murine melanoma cells

Retinoic acid induces growth arrest and differentiation in B16 mouse melanoma cells. Using gene arrays, we identified several early response genes whose expression is altered by retinoic acid. One of the genes, tbx2, is a member of T-box nuclear binding proteins that are important morphogens in developing embryos. Increased TBX2 mRNA is seen within 2 h after addition of retinoic acid to B16 cells. The effect of retinoic acid on gene expression is direct since it does not require any new protein synthesis. We identified a degenerate retinoic acid response element (RARE) between -186 and -163 in the promoter region of the tbx2 gene. A synthetic oligonucleotide spanning this region was able to drive increased expression of a luciferase reporter gene in response to retinoic acid; however, this induction was lost when a point mutation was introduced into the RARE. This oligonucleotide also specifically bound RAR in nuclear extracts from B16 cells. TBX2 expression and its induction by retinoic acid was also observed in normal human and nonmalignant mouse melanocytes.     

Maternal smoking and the retinoid pathway in the developing lung

Background

Maternal smoking is a risk factor for pediatric lung disease, including asthma. Animal models suggest that maternal smoking causes defective alveolarization in the offspring. Retinoic acid signaling modulates both lung development and postnatal immune function. Thus, abnormalities in this pathway could mediate maternal smoking effects. We tested whether maternal smoking disrupts retinoic acid pathway expression and functioning in a murine model.

Methods

Female C57Bl/6 mice with/without mainstream cigarette smoke exposure (3 research cigarettes a day, 5 days a week) were mated to nonsmoking males. Cigarette smoke exposure continued throughout the pregnancy and after parturition. Lung tissue from the offspring was examined by mean linear intercept analysis and by quantitative PCR. Cell culture experiments using the type II cell-like cell line, A549, tested whether lipid-soluble cigarette smoke components affected binding and activation of retinoic acid response elements in vitro.

Results

Compared to tobacco-naïve mice, juvenile mice with tobacco toxin exposure had significantly (P < 0.05) increased mean linear intercepts, consistent with an alveolarization defect. Tobacco toxin exposure significantly (P < 0.05) decreased mRNA and protein expression of retinoic acid signaling pathway elements, including retinoic acid receptor alpha and retinoic acid receptor beta, with the greatest number of changes observed between postnatal days 3–5. Lipid-soluble cigarette smoke components significantly (P < 0.05) decreased retinoic acid-induced binding and activation of the retinoic acid receptor response element in A549 cells.

Conclusions

A murine model of maternal cigarette smoking causes abnormal alveolarization in association with altered retinoic acid pathway element expression in the offspring. An in vitro cell culture model shows that lipid-soluble components of cigarette smoke decrease retinoic acid response element activation. It is feasible that disruption of retinoic acid signaling contributes to the pediatric lung dysfunction caused by maternal smoking.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

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.     

孤生受体的相互作用对视黄酸应答元件的影响

孤生受体ＣＯＵＰ－ＴＦ和ｎｕｒ７７的功能及其作用机理仍未阐明．以ＤＮＡ瞬时转染和测定氯霉素乙酰转移酶（ＣＡＴ）活性,以及凝胶阻抑测定,分析ＣＯＵＰ－ＴＦ和ｎｕｒ７７的相互作用对视黄酸应答元件（ＲＡＲＥｓ）的影响．实验表明,ＣＯＵＰ－ＴＦ通过降低ＲＡＲＥｓ的基础活性,来增强ＲＡＲＥ对视黄酸（ＲＡ）的敏感性,而ｎｕｒ７７则拮抗ＣＯＵＰ－ＴＦ的作用．ｎｕｒ７７能够加强不同ＲＡＲＥｓ的转录活性,并且与ＲＡ的诱导无关．结果证实,ｎｕｒ７７通过与ＣＯＵＰ－ＴＦ的直接作用而对ＲＡＲＥｓ产生影响,从而抑制ＣＯＵＰ－ＴＦ与ＲＡＲＥｓ结合和ＣＯＵＰ－ＴＦ的转录活性     

The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae

1988. The response of young Romney lambs to immunization with Trichostrongylus colubriformis larvae. International Journal for Parasitology 18: 1035–1038. Groups of weaned Romney ewe lambs were immunized with two doses of 28,000, 35,000 or 42,000 (2000 kg⁻¹) infective larvae of Trichostrongylus colubriformis at 8, 12 or 16 weeks of age, respectively. Each group and helminthfree control lambs of similar age were challenged with T. colubriformis at the same dose rate as used for immunization. Faecal egg counts (FEC) and haematological observations were made during the experiment, and at slaughter, 42 days after challenge, worm burdens were determined and small intestinal histology was examined.

Lambs in each immunized group were identified as ‘responders’ or ‘non-responders’ on the basis of both FEC and worm burdens. A significant (P<0.001) decrease in the worm burdens recovered as a proportion of the challenge infections in both unimmunized and immunized lambs with increasing age was observed.

Globule leukocyte numbers increased with the age of lambs. In addition, within each age group globule leukocyte numbers reflected individual responsiveness to immunization, significantly (P<0.01) greater numbers being present in ‘responders’ than ‘non-responders’ or unimmunized lambs. No difference in haematological responses were found in relation to the lambs' responsiveness to immunization.     

Different architectures of the collagen fibril: morphological aspects and functional implications

Several tissues known to contain collagen fibrils with a ‘helical’ arrangement were studied by t.e.m. and freeze-fracture. In all the tissues examined, the diameter of the collagen fibrils appeared to be tissue-specific and fairly constant within the same tissue. No statistical differences, on the contrary, were detectable in the coiling angle which appeared similar in all the tissues and independent of both diameter and age of the fibril. Rat tail tendon was also examined under the same technical conditions and showed collagen fibrils of large and very heterogeneous diameter and with a consistent ‘straight’ arrangement. These data seem to suggest that the ‘helical’ and ‘straight’ arrangements may actually identify different types of collagen fibrils. The authors discuss the possible functional significance of these arrangements and present two hypotheses on the three-dimensional structure of the ‘helical’ fibril.     

Retinoic Acid Metabolism Inhibition by 3-Azolylmethyl-1H-indoles and 2, 3 or 5-(α-Azolylbenzyl)-1H-indoles

Among a library of 70 azoles, 8 indole derivatives substituted in the 2-, 3- or 5- position with an azolylmethyl or α-azolylbenzyl chain were evaluated for retinoic acid (RA) metabolism inhibitory activity. The most active inhibitors identified in this study were 5-bromo-1-ethyl-3-methyl-2-[(phenyl)(1H-1,2,4-triazol-1-yl)methyl]-1H-indole (3) (68.9% inhibition) and 5-bromo-1-ethyl-2-[(4-fluorophenyl)(1H-1,2,4-triazol-1-yl)methyl]-3-methyl-1H-indole (6) (60.4% inhibition). At the same concentration (100 μM) ketoconazole exerted similar inhibitory effect (70% inhibition).     

Effect of epiphytes on the extent of necrotic injuries of resistant and susceptible poplar clones infected with Dothichiza populea.

Poplar cuttings of a resistant clone, Populus ‘Grandis’, and susceptible clones, Populus nigra ‘Italica’ and Populus ‘Robusta’, were infected with the pathogenic fungus Dothichiza populea alone, or with the pathogen and one of five strains of epiphytes antagonistic towards it (in vitro), isolated from poplar bark. The extent of injury was examined for 28 days after infection by determining the length of necrotic patches and their area as expressed in per cent of the total area of a cutting or the area of necrotic injuries caused by the pathogen alone.

All the poplar cuttings of both the resistant and susceptible clones became diseased when infected with the pathogen alone. Surprisingly enough, however, the least affected clone was the susceptible P. ‘Robusta’, in which necrotic injuries covered 28% of the total area, as against 40% and 70% in the resistant P. ‘Grandis’ and the susceptible P. nigra ‘Italica’, respectively.

When the cuttings were infected simultaneously with Dothichiza populea and its antagonistic epiphytes, the diseased area in the resistant clone diminished by as much as two-thirds, and in the susceptible P. nigra ‘Italica’, by one-third in comparison with the area affected by the pathogen alone. In turn, in the susceptible P. ‘Robusta’ the introduction of three out of five epiphytes stimulated the growth of the pathogenic fungus producing on average a double increase in the necrotic area. The differences in the response of the pathogen to the presence of epiphytes recorded in the susceptible clones indicate a marked influence of the plant on the nature of interactions between its epiphytic microflora and the pathogen.     

Intestinal absorption and metabolism of retinoyl beta-glucuronide in humans, and of 15-[14C]-retinoyl beta-glucuronide in rats of different vitamin A status

In order to prove the hypothesis that humans and animals with adequate vitamin A status do not absorb and metabolize orally administered all-trans retinoyl β-glucuronide, unlabeled retinoyl glucuronide (0.1 mmol) was orally dosed to fasting well-nourished young men. Neither retinoyl glucuronide nor retinoic acid, a possible metabolite, appeared in the blood within 12 h after ingestion. Next, radiolabeled all-trans 15-[¹⁴C]-retinoyl β-glucuronide was chemically synthesized by a new procedure, and fed orally to rats of different vitamin A status. Analysis of blood and other tissues 5 or 24 h after the dose, showed the presence of radioactivity ( 0.5%) in the blood of vitamin A deficient rats, but not in sufficient rats. Livers of all rats contained small, but detectable amounts (0.3 to 1.1% of the dose) of radioactivity. The accumulation of radioactivity in the liver was highest in deficient rats. Analysis of the retinoids showed that the radioactivity in serum and liver was due to retinoic acid formed from retinoyl glucuronide. Within 24 h after the dose, 31 to 40% of the administered radioactivity was excreted in the feces, and 2 to 4.7% of the dose was excreted in the urine. Results of the present studies show that oral administration of retinoyl β-glucuronide did not give rise to detectable changes in blood retinoyl glucuronide and/or retinoic acid concentrations in humans or rats with adequate vitamin A status.     

Viscoelastic properties of aqueous solutions of an exocellular polysaccharide from cyanobacteria

The viscoelastic properties of aqueous solutions of the exocellular polysaccharide of Cyanospira capsulata have been studied, over a wide range of polymer concentrations, using small deformation oscillatory, steady and transient shear methods. The viscoelastic spectra generally resemble those of an entangled network, although notable deviations can be observed in the low frequency dependence of G′ and G″. At higher polymer concentrations, the viscoelastic spectrum shows solid-like behaviour over a wide range of frequencies. The superposition of η^*(ω) and η( ) curves occurs only at low frequencies, at higher frequencies the slope of η^*(ω) is lower than that of η( ). By studying the time evolution of shear stress after the inception of a steady shear rate (stress overshoot), the recovery of non-linear properties after steady shearing flow is seen to occur after times of c. 10³ s (in the case of 1·1% w/v solutions).

The overall viscoelastic properties appear original in comparison with those of the two structurally limiting types of polysaccharide, the ‘ordered’ chain xanthan and the ‘random coil’ guar. A rationale for this ‘anomalous’ viscoelastic behaviour can be tentatively proposed in terms of flickering intermolecular cross-interactions between semi-flexible segments, which occur in addition to the usual topological constraints.     

Fatty acid composition in microspore-derived secondary embryos of Brassica napus L.

Fatty acid (FA) composition was determined in secondary embryos (SeEs) of Brassica napus L. of the cv. ‘Topas’ and ‘Janetzki’, and compared with the FA composition in microspore-derived embryos (MDEs) and seeds. SeEs of ‘Topas’, a rapessed cultivar with low erucic acid, revealed the same FA composition as observed in the MDEs. The FA pattern was, in most of the cases, independent of the procedures used for induction of SeEs e.g. low density cultures, transversal wounding, auxin treatment, and light conditions. However, non-embryogenic calli, as well as leaves from greenhouse- and in vitro-grown material had a FA composition distinctly different from the MDEs or SeEs. SeEs with an abnormal phenotype revealed differences in FA composition compared to MDEs or SeEs, but was distinct from the composition of non-embryogenic tissues. Virtually no erucic acid (22:1) or eicosaenoic acid (20:1) could be detected in SeEs of ‘Topas’. However, its presence in SeEs of ‘Janetzki’, a high erucic acid rapeseed cultivar, demonstrates that SeEs contain embryo-specific FAs. Under standard culture conditions, the levels of 22:1 and 20:1 were the same as observed in MDEs, which was approximately half the content observed in the seeds. The similarity in FA composition between SeEs and MDEs, of both low and high erucic acid rapeseed cultivars, is a unique characteristic which differs from most other somatic embryogenic systems.     

A new orphan member of the nuclear hormone receptor superfamily that interacts with a subset of retinoic acid response elements.

We have identified and characterized a new orphan member of the nuclear hormone receptor superfamily, called MB67, which is predominantly expressed in liver. MB67 binds and transactivates the retinoic acid response elements that control expression of the retinoic acid receptor beta 2 and alcohol dehydrogenase 3 genes, both of which consist of a direct repeat hexamers related to the consensus AGGTCA, separated by 5 bp. MB67 binds these elements as a heterodimer with the 9-cis-retinoic acid receptor, RXR. However, MB67 does not bind or activate other retinoic acid response elements with alternative hexamer arrangements or any of several other wild-type and synthetic hormone response elements examined. The transactivation of retinoic acid response elements by MB67 is weaker than that conferred by the retinoic acid receptors but does not require the presence of all-trans retinoic acid, 9-cis-retinoic acid, or any exogenously added ligand. We propose that MB67 plays an important role in the complex network of proteins that govern response to retinoic acid and its metabolites.     

A characteristic pharmacological action of ‘Yang-invigorating’ Chinese tonifying herbs: Enhancement of myocardial ATP-generation capacity

In order to investigate the pharmacological basis of ‘Yang-invigorating’ action, the effect of oral treatment with the methanolic extract of ‘Yang-invigorating’ herbs on ATP-generation capacity was examined, using heart homogenates prepared from herb-pretreated mice. Tonifying (i.e., health-promoting) herbs of other functional categories were also included for comparison. The results indicated that ‘Yang-invigorating’ Chinese tonifying herbs could invariably enhance myocardial ATP-generation capacity, with the extent of stimulation varying among the herbs. In contrast, ‘Yin-nourishing’ herbs either did not stimulate or even decreased myocardial ATP-generation capacity. While ‘Qi-invigorating’ herbs produced variable effects on myocardial ATP-generation capacity, most of the ‘blood-enriching’ herbs did not cause any significant changes. The results obtained from studies using myocardial mitochondrial fractions isolated from herb-pretreated mice suggest that ‘Yang-invigorating’ herbs might speed up ATP generation by increasing mitochondrial electron transport. The ensemble of results has provided evidence for the first time to support the pharmacological basis of ‘Yang invigoration’ in Chinese medicine.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.