Presence Of Retinol Research Articles

A Flow Injection Chemiluminescence Method for the Determination of Retinol in Pharmaceutical Formulations by Using Luminol-Diperiodatoargentate(III) Reaction.

In this paper, a chemiluminescence (CL) method is proposed for retinol determination by combining flow injection (FI) methodology. The CL reaction is based on the oxidation of luminol by diperiodatoargentate(III) (DPA) in the presence of retinol. Under the optimum conditions, the relative CL intensity was linear to the concentration of retinol over the range 5.0×10-3-14 mg L-1 (y=347.26x+2.5944, R2=0.9999, n=8) with limit of detection (LOD) of 1.5×10-3 mg L-1 (S/N=3) and limit of quantification (LOQ) of 5.0×10-3 mg L-1 (S/N=10). The relative standard deviation (RSD) was from 1.04-3.4% over the range studied and injection throughputs of 150 h-1. The method was satisfactorily applied to retinol in pharmaceutical formulation samples. The samples were saponified and extracted with liquid-liquid extraction using ether as an extractant. The possible CL mechanism is supported by CL and UV-visible spectrophotometric studies.

Retinol binding protein IV purified from Escherichia coli using intein-mediated cleavage as a suitable replacement for serum sources

Retinol binding protein IV (RBP) functions as the principal carrier of retinol (Vitamin A) in the blood, where RBP circulates bound to another serum protein, transthyretin. Isolation of pure RBP from the transthyretin complex in human serum can be difficult, but expression of RBP in recombinant systems can circumvent these purification issues. Human recombinant RBP has previously been successfully expressed and purified from E. coli, but recovery of active protein typically requires extensive processing steps, such as denaturing and refolding, and complex purification steps, such as multi-modal chromatography. Furthermore, these methods produce recombinant proteins, often tagged, that display different functional and structural characteristics across systems. In this work, we optimized downstream processing by use of an intein-based expression system in E. coli to produce tag-free, human recombinant RBP (rRBP) with intact native amino termini at yields of up to ~15 mg/L off column. The novel method requires solubilization of inclusion bodies and subsequent oxidative refolding in the presence of retinol, but importantly allows for one-step chromatographic purification that yields high purity rRBP with no N-terminal Met or other tag. Previously reported purification methods typically require two or more chromatographic separation steps to recover tag-free rRBP. Given the interest in mechanistic understanding of RBP transport of retinol in health and disease, we characterized our purified product extensively to confirm rRBP is both structurally and functionally a suitable replacement for serum-derived RBP.

Antioxidant and developmental capacity of retinol on the in vitro culture of rabbit embryos.

SummaryOxidative stress is a major cause of defective embryo development during in vitro culture. Retinoids are recognized as non-enzymatic antioxidants and may have an important role in the regulation of cell differentiation and vertebrate development. However, there are not enough reports discussing the antioxidant and developmental capacity of retinoids, including retinol (RT), on the in vitro development of embryos recovered from livestock animals, particularly in rabbit species. Therefore, morula embryos obtained from nulliparous Red Baladi rabbit does were cultured for 48 h in TCM199 medium in the absence of RT (control group) or in the presence of RT at concentrations of 10, 100 and 1000 nM. The developmental capacity to the hatched blastocyst stage, the antioxidant biomarker assay and the expression of several selected genes were analyzed in each RT group. The data show that RT significantly (P<0.001) promoted the embryo hatchability rate at the concentration of 1000 nM to 69.44% versus 29.71% for the control. The activity of malondialdehyde (MDA) level was significantly (P<0.05) lower in the RT groups than in the control group, while the total antioxidant capacity (TAC), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were significantly (P<0.05) higher following treatment with RT. Furthermore, RT treatment considerably upregulated the relative expression of gap junction protein alpha 1 (GJA1), POU class 5 homeobox 1 (POU5F1) and superoxide dismutase 1 (SOD1) genes compared with the control group. The current study highlights the potential effects of RT as antioxidant in the culture medium on the in vitro development of rabbit embryos.

Murine Embryonic Stem Cells Synthesize Retinoic Acid to Promote their Own Differentiation

Objective:Embryonic stem cells (ESC) pluripotency is maintained through a complex interplay of different signaling pathways and a network of transcription factors. Although much is known about this pluripotency selfrenewal circuitry, the molecular events that lead ESC to exit from pluripotency and begin differentiation are currently less known. All-trans retinoic acid (atRA), plays important and pleiotropic roles in embryonic development and ESC differentiation. The objective of the present study is to check the autocrine synthesis of atRA by ESC, test its role in spontaneous differentiation of ESC as embryoid bodies (EBs), and analyze the expression of the enzymes and proteins involved in atRA synthesis pathway. Methods: ESC, undifferentiated or differentiating as EBs, were grown in absence or presence of atRA, retinol, or the RAR antagonist CD2665, and Brachyury expression was analyzed as marker of ESC differentiation state. ESC or EBs-conditioned medium was produced in absence or presence of retinol and tested on RARE-luciferase reporter cells. RT-qPCR analysis of atRA biosynthetic pathway components was performed on undifferentiated or differentiating ESC. Finally, microarray gene expression profile was used to identify direct atRA target genes in ESC. Results: Here, we demonstrate that atRA promotes early steps of ESC differentiation, and that ESC increase their capacity to synthesize atRA during spontaneous differentiation as EBs, up-regulating RDH1, RDH10, ADH3, RALDH2, and CRABP2. Among 35 transcription factors (TFs) regulated by atRA in ESC, 3 TFs up-regulated (Snai1, Gata6, Cdx1) are known to be involved in ESC pluripotency exit and 3 TFs down-regulated (Otx2, Id2 and Arid1a) are involved in ESC pluripotency maintaining. Conclusion: Cultivation and controlled differentiation of ESC has opened new frontiers both in regenerative medicine and biology of development. Here, we demonstrated that RA is synthesized by ESC during spontaneous differentiation as EBs and takes an active role to promote their own differentiation process.

Analyses of the Secondary Particle Radiation and the DNA Damage It Causes to Human Keratinocytes

High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

High performance liquid chromatography method for the simultaneous determination of α-, γ- and δ-tocopherol in vegetable oils in presence of hexadecyltrimethylammonium bromide/n-propanol in mobile phase

A rapid methodology by RP-HPLC, 10min of total analysis time, for the analysis of α-, γ- and δ-tocopherol in vegetable oils, in presence of retinol, retinyl acetate and α-tocopherol acetate was developed. Hexadecyltrimethylammonium bromide 0.1M/n-propanol 65% (v/v) are used as mobile phase. Tocopherols were detected using UV and fluorescence detection. The quantification limits for α-, γ- and δ-tocopherols were 0.28, 0.12 and 0.23mgL−1, respectively. The method yielded satisfactory results when it was applied to vegetable oils and thus, it is suitable for their quality control.

Synergism between mefloquine and artemisinin and its enhancement by retinol in Plasmodium falciparum in vitro

Following the advent of mefloquine resistance in Plasmodium falciparum in Thailand in the 1990s, the combined treatment of falciparum malaria with artesunate and mefloquine was found to be highly effective in treating and curing the patients in the affected areas. Monitoring of the clinical-parasitological response and of the in vitro sensitivity of P. falciparum was systematically conducted in order to detect any signs of failure of this type of artemisinin-based combination treatment (ACT). In earlier observations the in vitro activity of artemisinin was found to be significantly enhanced when combined with retinol. The same applies to mefloquine. In order to check whether the synergism between artemisinin and mefloquine was maintained in the presence of retinol, the pharmacodynamic interaction of the three compounds was investigated in the western border area of Thailand. Successful parallel tests with mefloquine, artemisinin, retinol, mefloquine-artemisinin 5:1 as well as mefloquine-artemisinin (5:1) + retinol low, medium and high were obtained with 43 fresh parasite isolates. The retinol concentrations in the low, medium and high formulations corresponded to the 50th, 65th and 80th percentile of the physiological mean concentrations in the blood of healthy adults. The IC(50), IC(90) and IC(99) values for mefloquine alone showed a further increase over the data of 2008. In the combinations with artemisinin and retinol moderate synergism was observed at the IC(50), but synergism increased strongly at the IC(90) and the IC(99).

Flow injection methods for the determination of retinol and α‐tocopherol using lucigenin‐enhanced chemiluminescence

Flow injection (FI) methods are reported to determine retinol and α-tocopherol based on its enhancement affect of lucigenin chemiluminescence (CL) in alkaline medium. Surfactants including Brij-35, Triton X-100, cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate have been reported for the first time to enhance lucigenin CL intensity in the presence of retinol and α-tocopherol. With Brij-35, the CL intensity was enhanced by 67% for retinol and 58% for α-tocopherol. CTAB was found to enhance the CL intensity by 16% for retinol whereas for α-tocopherol, the CL intensity was quenched up to 95%. Retinol could be determined specifically in the presence of α-tocopherol using CTAB. The calibration graphs were found to be linear up to 1.43 mg/L (R(2) = 0.9985, n = 8) with a detection limit (3s) of 1.43 × 10(-3) mg/L for retinol and 2.15 mg/L (R(2) = 0.9989; n = 8) with a detection limit (3s) of 4.31 × 10(-4) mg/L for α-tocopherol. An injection throughput of 120/h, and relative standard deviations of 0.9-2.8% (n = 4) were achieved in the concentration range studied. The influence of common ions, excipients in pharmaceutical formulations and related organic compounds on the determination of retinol and α-tocopherol individually was studied. The proposed methods were applied to determine retinol and α-tocopherol in pharmaceutical formulations and human blood serum. The results did not differ significantly from the CL method and HPLC reference method at 95% confidence level.

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development #

The mesothelial cells which are desmin-positive but negative for smooth muscle actin-alpha (SMAα). The perivascular mesenchymal cells which are desmin and SMA-positive with single desmin-positive SMA-negative cells. The parenchymal mesenchymal cells (hepatic stellate cells [HSCs]) which are desmin-positive and SMAα-negative with single exceptions (SMAα-positive and desmin-positive). Could the single desmin-positive and SMAα-positive cells of the parenchyma (in Fig. 2E,F of Asahina et al.) represent the cells of the arterial capillary of the sinusoids? Did the enriched HSC population contain SMAα RNA? Is it possible that the desmin-positive, SMAα-negative cells (HSCs) do not survive in culture? If they survive (ALCAM-high) acquiring the myofibroblastic phenotype, does the SMAα gene expression decrease when they are cultured on collagen in the presence of retinol and palmitic acid, and does ALCAM remain positive (immunocytology)? Is it possible to make similar experiments using enriched HSCs? Giuliano Ramadori M.D.*, Tümen Mansuroglu M.D.*, * Department of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg-August-University Göttingen, Göttingen, Germany.

Synthesis and properties of polyaniline nanolayers in the presence of retinol in aqueous ethanol

AbstractPolyaniline (PANI) nanolayers were shown to form spontaneously during the chemical oxidation in the presence of retinol using pure water and aqueous ethanol as reaction media. The effects of the retinol, the volume fraction of ethanol, and the acidity of the polymerization medium on the D.C. conductivity of PANI were investigated through two‐probe method at room temperature. The introduction of 0.2 g retinol improved the D.C. conductivity value of PANI to 3.0 S/cm in pure water and 4.46 S/cm in 50% aqueous ethanol when the H+ concentration was 0.5M. The D.C. conductivity values were 5.1 and 5.49 S/cm when the concentration of H+ was 1.0M in pure water and 2.5M in 50% aqueous ethanol, respectively. The products were also characterized by Fourier transform infrared spectroscopy, ultraviolet–visible spectroscopy, scanning electron microscope, cyclic voltammetry, and X‐ray diffraction techniques. Moreover, the resulting PANI nanolayers exhibited an unusual electromagnetic loss at the microwave frequency (f = 8.2–12.4 GHz) and it arose from order arrangement of polaron as charge carrier caused by nanolayers morphologies and the materials can be used for the potential application as microwave absorbing materials. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Retinol binding protein is a major carrier of progesterone in vitro and in human follicular fluid

OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands.DESIGN: Experimental study.MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF.RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex.CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells. OBJECTIVE: The human retinol binding protein 4 (RBP) is a member of the lipocalin family of proteins and is the specific vitamin A carrier. It has diverse biological functions including intra- and extracellular transport of retinoids, regulation of reproduction and insulin resistance signaling. Our recent proteomic analysis of human follicular fluid (FF) demonstrate high level of RBP presence and nearly undetectable level of retinoids. This study was conducted to investigate the RBP function in human FF as a possible carrier of other than retinals hydrophobic ligands. DESIGN: Experimental study. MATERIALS AND METHODS: Recombinant human RBP (rhRBP) was isolated from bacteria as inclusion bodies and purified by HPLC. Renaturation was performed by dialysis in the presence of retinol, cholesterol, or progesterone as ligands. rhRBP complexation with the ligands was verified by HPLC, ELISA, and immunoprecipitation with RBP-specific antibody, A/G Sepharose, and Western blot. Presence of retinol and progesterone in the complexes was monitored through 3H-labeled ligands as a trace. Complexes were formed also with purified TTR. rhRBP-ligand and rhRBP-TTR-ligand complexes (0.1–0.4uM) were added to primary human ovarian fibroblast cell culture (FC). Ligand delivery to FC was monitored by measuring cellular 3H-ligand incorporation after 3h incubation. FF and FC were obtained at the time of vaginal oocyte retrieval from IVF patients (3 young donor patients, age <28 years) and cultured in DMEM medium. Fetal calf serum was omitted during the rhRBP-ligand and rhRBP-TTR-ligand incubation. HPLC and immunocapture were used to search for RBP-progesterone and RBP–cholesterol complexes in FF. RESULTS: We confirmed 3H-Progesterone-rhRBP and 3H-Progesterone-rhRBP-TTR complexation using purified (>99% purity) recombinant proteins. The binding affinity of progesterone-rhRBP complexation is similar to the binding affinity of the retinol-rhRBP binding. Cholesterol was found to bind rRBP and rhRBP-TTR with very low affinity (<3%). >40% of the progesterone from the progesterone-rhRBP complex was found to enter FC. The presence of TTR did not change dramatically progesterone uptake. More than 60% of the FF progesterone is present as a constituent of the RBP-TTR complex. CONCLUSIONS: rhRBP alone and in a complex with TTR is a progesterone but not cholesterol carrier. In FF, RBP-TTR is a major carrier of progesterone. In a complex with RBP or RBP-TTR progesterone is delivered into the cells.

Changes in Structure and in Interactions of Heat-Treated Bovine &amp;#946;-Lactoglobulin

Heat stress on structure and ligand binding of beta-LG has been studied by fluorescence, circular dichroism and gel electrophoresis at pH 6.5. Native PAGE gel electrophoresis shows that denaturation of beta-LG is reversible up to 75 degrees C then it becomes irreversible due to aggregation of beta-LG. Formation of aggregated beta-LG is completed at 95 degrees C. Circular dichroism results indicate that formation of aggregated beta-LG is accompanied by the scrambling of disulfide bonds (creation of new intramolecular and intermolecular disulfide bridges and rearrangement of old intramolecular disulfide bridges). Addition of ethanolic retinol causes a change in polarity of the solution and favors transformation of the beta<-->alpha structure. In the presence of retinol, the alpha-helix content of the secondary structure of heat-treated beta-LG is increased and the major portion of its secondary structure is helical. Fluorescence results show that heat-treated beta-LG at 95 degrees C can still bind retinol. The refolding of the tertiary structure of beta-LG heat-denatured at 95 degrees C may recreate a retinol binding site. Surprisingly, the affinity of the new site for retinol is higher than that of native beta-LG; however, the apparent molar ratio is lower than one. The binding properties of beta-LG for terpenoids have been measured after its heat treatment at 20, 75 and 95 degrees C. The intensity of tryptophan emission at 330 nm was changed only in the case of the interaction with beta-ionone. Other ligands probably cannot bind to beta-LG or they bind in a binding site far from the tryptophan residues, hence not affecting its fluorescence.

Profile and regulation of annexin II expression during early embryogenesis in cattle

The presence of annexin II (Ann-II) during the initial stages of bovine embryo development and the regulation of Ann-II expression by retinol and insulin-like growth factor I (IGF-I) were studied. Bovine embryos at different stages of development were produced in vitro on Synthetic Oviductal Fluid (SOF) medium (control group), SOF supplemented with retinol (retinol group; 0.1ng/ml), or IGF-I (IGF-I group; 10ng/ml). The embryos were processed for mRNA extraction, cDNA production and polymerase chain reaction (PCR) using Ann-II-specific oligonucleotides. Ann-II was detected in all stages of early embryo development, except for the 16-cell stage. The blastocyst rates were significantly higher (P&lt;0.05) in the group supplemented with retinol (37.8%, 45/119) during in vitro embryo culture (IVC) than in those cultured in SOF (20.5%, 24/117) or SOF with IGF-I (25.8%, 24/93). Semiquantitative analysis of Ann-II expression in embryos produced in medium supplemented with IGF-I or retinol revealed a lower expression of this gene when compared with embryos cultured in SOF (P&lt;0.05). The Ann-II expression was not different in embryos cultured in the presence of retinol and IGF-I. The presence of retinol increased the production of embryos in vitro by decreasing the expression of Ann-II in early-stage of bovine embryo.

α-Tocopherol increases caspase-3 up-regulation and apoptosis by β-carotene cleavage products in human neutrophils

It has been found that β-carotene cleavage products (CarCP), besides having mutagenic and toxic effects on mitochondria due to their prooxidative properties, also initiate spontaneous apoptosis of human neutrophils. Therefore, it was expected that antioxidants such as α-tocopherol would inhibit the stimulation of apoptosis and caspase-3 activity by CarCP. However, we found that α-tocopherol increases caspase-3 up-regulation and stimulation of apoptosis of human neutrophils by CarCP. Ascorbic acid does not alter this caspase-3 up-regulating and proapoptotic effect exerted by α-tocopherol. Both α-tocopherol and ascorbic acid, in the absence of CarCP, decrease intracellular caspase-3 activity and spontaneous apoptosis of neutrophils. Uric acid alone or in combination with CarCP does not exert apparent effects on caspase-3 activity and apoptosis. Up-regulating effect of α-tocopherol is not observed in the presence of retinol that markedly stimulates apoptosis by itself, whereas increase of caspase-3 activity is induced by concomitant addition of α-tocopherol and β-ionone, a cyclohexenyl degradation product of β-carotene with shorter aliphatic chain.

Cell proliferation inhibition and alterations in retinol esterification induced by phytanic acid and docosahexaenoic acid

We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not. PA and DHA treatment increased the levels of retinyl esters (REs) in both PrECs and PC-3 cells and generated novel REs that eluted on reverse-phase HPLC at 54.0 and 50.5 min, respectively. Mass spectrometric analyses demonstrated that these novel REs were retinyl phytanate (54.0 min) and retinyl docosahexaenoate (50.5 min). Neither PA nor DHA increased LRAT mRNA levels in these cells. In addition, we demonstrate that retinyl phytanate was generated by LRAT in the presence of PA and retinol; however, retinyl docosahexaenoate was produced by another enzyme in the presence of DHA and retinol.

Annexin II mRNA expression in bovine oocytes during follicular development

We investigated the expression of calcium-dependent phospholipid binding protein annexin-II (Ann-II) messenger RNA (mRNA) during preantral follicle development and in oocytes from antral follicles of different diameters (< 3 mm, 5 to 8 mm and > 8 mm). The action of retinol on Ann-II mRNA expression in mature oocytes was also examined. Only oocytes from secondary preantral follicles expressed Ann-II mRNA and at the germinal vesicle stage expression by oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) compared with oocytes from follicles smaller than 3 mm or between 5 and 8 mm. Ann-II mRNA expression by metaphase II oocytes from follicles larger than 8 mm was significantly higher (p < 0.05) than that from oocytes from follicles smaller than 3 mm, with oocytes from both these size-classes showing similar levels of Ann-II mRNA expression as oocytes recovered from 5-8 mm follicles. In the presence of retinol, Ann-II mRNA expression was higher than when retinol was absent (p < 0.05). Our data indicate that Ann-II mRNA expression is highest in competent oocytes and that retinol increases Ann-II mRNA and may be involved in the regulation of oocyte competence by decreasing the translation and/or degradation of Ann-II mRNA.

Action of thiazolidinediones on differentiation, proliferation and apoptosis of normal and transformed thyrocytes in culture

Differentiating drugs may be able to re-sensitize thyroid carcinomas to radioiodine therapy. Substituted thiazolidinediones (TZDs) belong to the group of oral anti-diabetic drugs that also possess anti-proliferative and pro-apoptotic effects and, potentially, differentiating effects on several cancer cell lines. Some of the effects are mediated via the peroxisome proliferator-activated receptor gamma (PPAR-gamma). We investigated the effect of troglitazone, rosiglitazone and pioglitazone on differentiation in normal porcine thyrocytes and in the follicular carcinoma cell lines FTC 133 and FTC 238. Differentiation was investigated by measuring 125I uptake and the expression of sodium-iodide symporter and thyroglobulin proteins. The TZDs were tested in the presence of retinol and retinoic acid. Additionally, proliferation was evaluated by [3H]thymidine uptake and cell number and apoptosis by annexin V-labeling. Controls included tocopherol and unsubstituted thiazolidinedione and co-incubation of the TZDs with the PPAR-gamma antagonist GW9662. PPAR-gamma and retinol X receptor (RXR)-alpha were investigated by immunocytochemistry, Western blot and RT-PCR. Cells derived from the metastasis showed greater responses than cells derived from the primary tumor. Troglitazone showed greater effects than the other TZDs. Troglitazone significantly increased 125I uptake and apoptosis and decreased [3H]thymidine uptake and cell number. The amount of the sodium iodide-symporter in the membrane fraction was significantly increased, while that of thyroglobulin was not influenced by the treatment. Inclusion of antagonist did not abolish these effects. No synergistic effect with any retinoid was detected. All transformed cells expressed PPAR-gamma and RXR-alpha but TZDs did not change their expression. Troglitazone appears to be suited for the re-differentiation treatment of dedifferentiated thyroid carcinoma because its action is twofold. On the one hand it increases differentiation and on the other hand it inhibits proliferation.

Solvent-induced ligand dissociation and conformational states of Cellular Retinol-Binding Protein Type I

Cellular Retinol-Binding Protein type I (CRBP) exhibits very high affinity for its ligand, bound within a buried cavity completely shielded from the outside medium. Three-dimensional structure and backbone dynamics in aqueous solution at neutral pH, either in the absence or in the presence of retinol, fail to represent the protein in a state capable of ligand uptake and release. The question was asked whether changes in the composition of the outside medium might facilitate ligand dissociation. Acidic aqueous solutions and water–alcohol mixtures were selected, among the best described denaturing solvents, to investigate their effects on the stability of the carrier–ligand complex and the conformational state of the protein upon ligand release. Circular dichroism (CD) and fluorescence spectroscopy were used to probe protein secondary and tertiary structure, compactness and retinol dissociation. While in purely aqueous media retinol dissociation parallels the acid-induced denaturation of the carrier, in water–alcohol mixtures it occurs in a range of co-solvent content lower than that required for protein denaturation. In light of these results, it is suggested that local solvent properties in vivo might modulate protein conformation and flexibility and thus play a fundamental role in the control of retinol exchange between carrier and membrane-bound donors and acceptors.

Vitamin E activates CRABP-II gene expression in cultured human fibroblasts, role of protein kinase C

The treatment of human fibroblasts with different tocopherols in the presence of retinol caused an increase in cytoplasmic retinoic acid binding protein II (CRABP-II) mRNA and protein. The possibility of an involvement of protein kinase C (PKC) in the response to tocopherols was supported by the results obtained with the PKC-specific inhibitors, calphostin C and bisindolylmaleimide I. The effect of α-tocopherol was prevented by okadaic acid, suggesting that a protein phosphatase is responsible for PKC dephosphorylation produced by the presence of tocopherols. The results shown support the hypothesis that phosphorylation/dephosphorylation of RXRα via PKC may be involved in the regulation of CRABP-II gene expression.

Effect of pentoxifylline on arachidonic acid metabolism, neutral lipid synthesis and accumulation during induction of the lipocyte phenotype by retinol in murine hepatic stellate cell.

In liver fibrosis, the quiescent hepatic stellate cells (HSC) are activated to proliferate and express the activated myofibroblast phenotype, losing fat droplets and the stored vitamin A, and depositing more extracellular matrix. Therapeutic strategies for liver fibrosis are focused on HSC. Pentoxifylline (PTF), an analog of the methylxanthine, prevents the biochemical and histological changes associated with animal liver fibrosis. The aim of the present study was to investigate the phenotypic change of myofibroblasts into quiescent lipocytes by PTF and/or retinol, using a permanent cell line GRX that represents murine HSC. We studied the action of both drugs on the synthesis of neutral lipids, activity of phospholipase A2 (PLA2), release of arachidonic acid (AA) and prostaglandins synthesis. Accumulation and synthesis of neutral lipids was dependent upon association of retinol with PTF. PTF (0.5 mg/mL) alone did not induce lipid accumulation and synthesis, but in cells induced by physiologic concentration of retinol (1-2.5 microM), it increased the quantity of stored lipids. Retinol and PTF (5 microM and 0.1 mg/mL, respectively) had a synergistic effect on neutral lipid synthesis and accumulation. In higher PTF concentrations (0.5 and 0.7 mg/ml), the synthesis was stimulated but accumulation decreased. Membrane-associated PLA2 activity decreased after PTF treatment, which increased the AA release 8 fold, and significantly increased the production of PGE2, but not of PGF2. However, when in presence of retinol, we observed a slightly higher increase in PGE2 and PGF2a production. In conclusion, PTF treatment generated an excess of free AA. We propose that retinol counteracts the action of PTF on the AA release and PGs production, even though both drugs stimulated the lipocyte induction in the HSC.