Effects of indole‐3‐butyric acid on growth,pigments and UV‐screening compounds in Nostoc linckia

In the present research, the effect of indole‐3‐butyric acid (IBA) on the growth, and the production of some primary and secondary metabolites was studied in Nostoc linckia. In this respect, algae cultures were supplied with 0, 0.01, 0.1, 1, 10, and 100 μM IBA for 14 days. IBA at concentrations of 10 and 100 μM induced algal growth expressed as fresh weight in N. linckia. Treatment with IBA at all concentrations stimulated heterocyst formation. In addition, low concentrations of IBA (0.01, 0.1, and 1 μM) had a stimulatory effect on chlorophyll a and carotenoids accumulation. In contrast, higher concentrations of IBA induced the accumulation of phycocyanin, allophycocyanin, and phycoerythrin in the treated algae. In this case, IBA at the concentration of 10 μM was more effective. A significant decrease in protein content was observed in the algae treated by 0.01 μM IBA. All concentrations of IBA caused a decrease in sugar content, but lower concentrations were more effective. IBA application in all of the concentrations except 100 μM increased oligosaccharide‐linked mycosporine‐like amino acids (OS‐MAAs) content. Lower concentrations had a more significant effect on increasing OS‐MAAs content. However the concentrations of 10 and 100 μM IBA decreased scytonemin content. These results indicated the stimulatory impact of IBA on weight, heterocyst formation, and photosynthetic pigments in N. linckia.     

Dickkopf‐3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells

Dickkopf‐3 (Dkk‐3) and Dkkl‐1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera‐2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk‐3 but not of Dkkl‐1. Ectopic expression of both Dkk‐3 and Dkkl‐1 induced apoptosis in NT2 cells. Gene silencing of Dkk‐3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk‐3‐silenced cells, this morphological response was not observed in Dkk‐3‐silenced cells. These findings suggest that Dkk‐3 plays a role in the regulation of cell interactions during RA‐induced neuronal differentiation. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1243–1254, 2014     

Regulation of Gdnf expression by retinoic acid in Sertoli cells

Glial cell line‐derived neurotrophic factor (GDNF) and retinoic acid (RA) are two molecules crucial for the regulation of the spermatogonial compartment of the testis. During the cycle of the seminiferous epithelium, their relative concentration oscillates with lower GDNF levels in stages where RA levels are high. It has been recently shown that RA negatively regulates Gdnf expression but the mechanisms behind are so far unknown. Here, we show that RA directly downregulates Gdnf mRNA levels in primary murine Sertoli cells through binding of RARα to a novel DR5‐RARE on Gdnf promoter. Pharmacological inhibition and chromatin immunoprecipitation–quantitative polymerase chain reaction analysis suggested that the underlying mechanism involved histone deacetylase activity and epigenetic repression of Gdnf promoter upon RA treatment.     

Up-regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter-response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid (RA) involve up-regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA-dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3-like motif that directly interacts with retinoid X receptor (RXR) alpha in a sequence-specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA-induced SOX3 gene expression could be significantly down-regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up-regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings.     

Characterization of the effects of butyric acid on cell proliferation, cell cycle distribution and apoptosis

We examined concentration-dependent changes in cell cycle distribution and cell cycle-related proteins induced by butyric acid. Butyric acid enhanced or suppressed the proliferation of Jurkat human T lymphocytes depending on concentration. A low concentration of butyric acid induced a massive increase in the number of cells in S and G2/M phases, whereas a high concentration significantly increased the accumulation of cells in G2/M phase, suppressed the accumulation of cells in G0/G1 and S phases, and induced apoptosis that cell cycle-related protein expression in Jurkat cells treated with high levels of butyric acid caused a marked decrease in cyclin A, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4 and CDK6 protein levels in G0/G1 and S phases, with apoptosis induction, and a decrease in cyclin B, Cdc25c and p27KIP1 protein levels, as well as an increase in p21CIP1/WAF1 protein level, in the G2/M phase. Taken together, our results indicate that butyric acid has bimodal effects on cell proliferation and survival. The inhibition of cell growth followed by the increase in apoptosis induced by high levels of butyric acid were related to an increase in cell death in G0/G1 and S phases, as well as G2/M arrest of cells. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Micro‐computed tomographic evaluation of fetal skeletal changes induced by all‐trans‐retinoic acid in rats and rabbits

BACKGROUND: Our laboratory has been conducting positive control studies to evaluate the utility of micro‐computed tomography (micro‐CT) for qualitative evaluation of fetal skeletal morphology. All‐trans‐retinoic acid (atRA) was used to produce a different spectrum of defects compared to our previous studies with boric acid and hydroxyurea. METHODS: Groups of five mated Crl:CD(SD) female rats each were administered vehicle or atRA (2.5–50 mg/kg) on GD 10, and groups of four mated Dutch Belted rabbits each were dosed with vehicle or atRA (6.25–25 mg/kg) on GD 9. Cesarean sections were performed on GD 21 and 28, respectively. Following external examination the viscera were removed and fetuses scanned in a micro‐CT imaging system. Fetuses were subsequently stained with alizarin red. Skeletal morphology was evaluated by each method without the knowledge of treatment group. Total bone mineral content (BMC) of each fetus was quantitated using the micro‐CT images. RESULTS: In rats there were dose‐related increases in the incidence of extra lumbar vertebra and non‐dose‐related increases in supernumerary ribs at all dose levels. There were decreases in mean number of ossified sacrocaudal vertebra at ≥5 mg/kg, and increases in skull bone malformations at ≥10 mg/kg. Rabbits were less sensitive on a mg/kg basis since skeletal malformations and a decrease in mean number of ossified sacrocaudal vertebra were observed only in the 25‐mg/kg group. Micro‐CT evaluation detected essentially the same incidence of skeletal abnormalities as seen in alizarin red‐stained rat and rabbit fetuses. BMC analysis showed a trend toward slight decreases in atRA‐treated rats, but no notable changes in rabbits. CONCLUSIONS: These results add support to our previous work that demonstrates that micro‐CT imaging can effectively assess rat and rabbit fetal skeletal morphology. Birth Defects Res (Part B) 89:408–417, 2010. © 2010 Wiley‐Liss, Inc.     

Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori)

Cellular retinoic acid binding protein (CRABP) is a member of intracellular lipid-binding protein (iLBP), and closely associated with retinoic acid (RA) activity. We have cloned the CRABP gene from silkworm pupae and studied the interaction between Bombyx mori CRABP (BmCRABP) and all-trans retinoic acid (atRA). The MTT assay data indicated that when BmCRABP is overexpressed in Bm5 cells, the cells dramatically resisted to atRA-induced growth inhibition. Conversely, the cells were sensitive to atRA treatment upon knocking down the BmCRABP expression. Subcellular localization revealed that BmCRABP is a cytoplasm protein, even when treated with atRA, the CRABP still remained in the cytoplasm. These data demonstrated that the function of BmCRABP have an effect on the physiological function of atRA.     

Potential signal pathway of all-trans retinoic acid for MMP-2 and MMP-9 expression in injury podocyte induced by adriamycin

Abstract

All-trans-retinoic acid (ATRA) can regulate some specific genes expression in various tissue and cells via nuclear retinoic acid receptors (RARs), including three subtypes: retinoic acid receptor-alpha (RAR-α), retinoic acid receptor-beta (RAR-β) and retinoic acid receptor-gamma (RAR-γ). Podocyte injury plays a pivotal role in the progression of glomerulosclerosis (GS). This study was performed to study the potential signal pathway of ATRA in the expression of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) in injury podocyte. Cells were divided into three groups: group of negative control (NC), group of injury podocyte induced by adriamycin (ADR) (AI) and group of ADR inducing podocyte injury model treated with ATRA (AA). The cells morphology changes were detected using microscope and scanning electron microscopy. MMP-2 and MMP-9 enzymic activity was detected using the gelatin zymography method. Protein and mRNA expressions of MMP-2, MMP-9, RAR-α, RAR-β and RAR-γ were measured by western-blot and real-time RT-PCR. Enzymatic activity of MMP-2 and MMP-9 in group AA was significantly enhanced compared to AI group after ATRA-treated 24?h (p?<?0.05). The protein and mRNA expressions of MMP-2/MMP-9 in group AA were significantly increased than those in group AI at both 12 and 24?h time points (p?<?0.05). Compared to group AI, RAR-α and RAR-γ protein/mRNA expressions of group AA were significantly increased at both 12 and 24?h time points (p?<?0.05). There was no difference for the expression of RAR-β between group AI and group AA (p?>?0.05). RAR-α protein level was positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05), and RAR-γ protein level was also positively correlated with MMP-2 or MMP-9 protein expression (p?<?0.05). In conclusion, ATRA may increase expression of MMP-2 and MMP-9 by the potential signal pathway of RAR-α and RAR-γ in injury podocyte induced by adriamycin, but not RAR-β.     

Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures

The rate of polarised secretion of sialosyl Lewis^a(19-9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 μM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewis^a levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine- to twentyfold increase in sialosyl Lewis^a epitope levels in a light membrane subfraction from RA-treated cells. Antigenic molecules of < 200,000 Mr on acrylamide gradient gels were concentrated in two doublets in the apparent Mr range 106,000–152,000 on Western blots. Carbohydrates analyses of oligosaccharides from SiaLeams of membrane subfraction and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewis^a, fucose/total CHO, and (³H) fucose incorporation in control samples than RA samples. Western blots of samples from membranes subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewis^a epitope, RA treatment did not augment cysteine-rich, PDTRP, blood group H-2, blood group A, and EGF receptor-like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewis^a induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Galβ1-3 moieties at oligosaccharide termini destined for export from the Golgi.     

Effects of 900 MHz electromagnetic fields exposure on cochlear cells' functionality in rats: evaluation of distortion product otoacoustic emissions

In recent years, the widespread use of mobile phones has been accompanied by public debate about possible adverse consequences on human health. The auditory system is a major target of exposure to electromagnetic fields (EMF) emitted by cellular telephones; the aim of this study was the evaluation of possible effects of cellular phone-like emissions on the functionality of rat's cochlea. Distortion Products OtoAcoustic Emission (DPOAE) amplitude was selected as cochlea's outer hair cells (OHC) status indicator. A number of protocols, including different frequencies (the lower ones in rat's cochlea sensitivity spectrum), intensities and periods of exposure, were used; tests were carried out before, during and after the period of treatment. No significant variation due to exposure to microwaves has been evidenced.     

Effects of retinoic acid metabolites on proliferation and differentiation of the clonal rhabdomyosarcoma cell line BA-HAN-1C

Summary— The clonal rat rhabdomyosarcoma cell line BA-HAN-IC is composed of proliferating mononuclear cells, some of which spontaneously fuse to terminally differentiated myotube-like giant cells. This cell line has been shown to be susceptible to differentiation induction with all-trans retinoic acid (RA). Since it is still unknown whether exclusively all-trans RA itself or also its metabolites can act as inductive compounds in our cell line, we exposed BA-HAN-1C cells to the metabolites 4-hydroxy RA, 4-oxo RA and 5,6epoxy RA. Exposure to these RA metabolites resulted in a significant inhibition of proliferation (P < 0.001) and induction of cellular differentiation, as evidenced by a significant increase in the number of myotube-like giant cells (P < 0.05) and a significant increase in creatine kinase activity (P < 0.05). However, differences in the inductive potency of these RA metabolites became apparent. Furthermore, RA metabolites exhibited a significantly weaker (P < 0.05) inductive activity when compared to all-trans RA. Summarizing our results we could demonstrate that the endogenous metabolites 4-hydroxy RA, 4-oxo RA and 5,6-epoxy RA are not merely deactivated cellular excretion products of all-trans RA, but potent inducers of differentiation and inhibitors of proliferation, possibly contributing to the complex physiological actions of retinoic acid.     

Effect of all-trans-retinoic acid on the expression of primordial germ cell differentiation-associated genes in mESC-derived EBs

atRA (all-trans-retinoic acid) is known to induce the differentiation of mESCs (mouse embryonic stem cells) into PGCs (primordial germ cells) in vitro. However, it is not clear as to what changes occur in PGC differentiation-associated genes or what mechanisms are involved when EBs (embryoid bodies) derived from mESCs are induced by atRA. EBs derived from mESCs were treated with 1, 2 or 5 μM atRA for 16 h, 2 days or 5 days. Real-time PCR and Western blot analysis were performed to detect the relative levels of PGC differentiation-associated genes (Lin28, Blimp1, Stra8 and Mvh) and the corresponding proteins respectively. Immunofluorescence was used to detect the protein location and distribution in EBs. The expression characteristics of genes could be divided into three categories: rapidly reached the peak value in 16 h and then decreased (Stra8, Lin28), initially low and then increased to reach the peak value in 5 days (Mvh) and relatively unchanged (Blimp1). A low level of Lin28 was expressed in EBs treated with atRA for 2 days or 5 days. The variation in the level of Lin28 mRNA did not influence the change in the level of Blimp1 mRNA. The changes in Stra8/Lin28 were consistent with the corresponding changes in the levels of their respective mRNAs, but the changes for Mvh/Blimp1 were not consistent with the corresponding changes in the levels of their respective mRNAs. Blimp1 expression may be independent of the effect of atRA on PGC differentiation. atRA may promote the start of a period in which there is a low level of Lin28 expression during PGC differentiation.     

Effect of adriamycin treatment on the lifetime of pyrene butyric acid in single living cells

We investigated the fluorescence lifetime of pyrene butyric acid (PBA) using various O₂ concentrations in cells. Both in living and freshly fixed cells, PBA lifetime decreased with oxygen concentration. We recorded decay curves in single cells and measured PBA lifetime and NAD(P)H intensity values. Under nitrogen atmosphere, the probe lifetime differences (199 and 209 ns in living and freshly fixed cells, respectively) suggest a supplemental pathway for the deactivation of the probe when the cell functions are not stopped. We propose reactive oxygen species (ROS) to be the additional quenchers that cause this decrease. We further studied the effect of drugs generating ROS the anthracycline doxorubicin (adriamycin). For living cells, PBA lifetime decreased after adriamycin (ADR) treatment (200 and 1000 ng/ml). This supports our hypothesis that under nitrogen atmosphere and for freshly fixed cells, PBA lifetimes increase to an unchanging value due to absence of quenchers.     

All‐trans retinoic acid upregulates the expression of ciliary neurotrophic factor in retinal pigment epithelial cells

Retinopathy of prematurity, a leading cause of visual impairment in low birth‐weight infants, remains a crucial therapeutic challenge. Ciliary neurotrophic factor (CNTF) is a promyelinating trophic factor that promotes rod and cone photoreceptor survival and cone outer segment regeneration in the degenerating retina. Ciliary neurotrophic factor expression is regulated by many factors such as all‐trans retinoic acid (ATRA). In this study, we found that ATRA increased CNTF expression in mouse retinal pigment epithelial (RPE) cells in a dose‐ and time‐dependent manner, and PKA signaling pathway is necessary for ATRA‐induced CNTF upregulation. Furthermore, we showed that ATRA promoted CNTF expression through CREB binding to its promoter region. In addition, CNTF levels were decreased in serum of retinopathy of prematurity children and in retinal tissue of oxygen‐induced retinopathy mice. In mouse RPE cells cultured with high oxygen, CNTF expression and secretion were decreased, but could be recovered after treatment with ATRA. In conclusion, our data suggest that ATRA administration upregulates CNTF expression in RPE cells.     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: Modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-β family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused ∼2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7–14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3–5-fold vs. 2–3-fold increase in ALP; ∼40% vs. ∼20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by ∼2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca²⁺ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo. J. Cell. Biochem. 67:498–513, 1997. © 1997 Wiley-Liss, Inc.     

Induction of apoptosis and change of bcl—2 expression in macrophage Ana—1 cells by all—trans retinoic acid

Macrophage cells play an important role in the initiation and regulation of the immune response.All-trans retinoic acid (ATRA) and its natural and synthetic analogs (retinoids)affect a large number of biological processes.Recently,retinoids have been shown promise in the therapy and prevention of various cancers.However,many interesting questions related to the activities of retinoids remain to be answered:(I) Molecular mechanisms by which retinoids exert their effects;（Ⅱ)why the clinical uses of retinoids give undesirable side effects of varying severity with a higher frequency of blood system symptoms;(Ⅲ)little is known for its impacts on macrophage cells etc.We set up this experiment,therefore,to examine the apoptosis of ATRA on macrophage Ana-1 cell line.Apoptosis of the cells was quantitated,after staining cells with propidium iodide(PI),by both accounting nuclear condensation and flow cytometry.When the cells were treated with ATRA at or higher than 1μM for more than 24h,significant amount of the apoptotic cells was observed.Induction of apoptosis of Ana-1 cells by ATRA was in time-and dose-dependent manners,exhibiting the similar pattern as the apoptosis induced by actinomycin D (ACTD).ATRA treatment of Ana-1 cells also caused the changes of the mRNA levels of apoptosis-associated gene bcl-2,as detected by Northern blot analysis.The temporal changes of bcl-2 expression by ATRA was also parallel to that by ACTD.In conclusion,ATRA can induce apoptosis in macrophage cells,which may be helpful in understanding of immunological functions retinoids.