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B Shroot

Publications and source records attributed to B Shroot.

At least 19 recordsLinked to original sources
PubMed1992

Identification of synthetic retinoids with selectivity for human nuclear retinoic acid receptor gamma.

The action of retinoids on gene regulation is mediated by three distinct nuclear retinoic acid receptor (RAR) subtypes called RAR alpha, beta and gamma. Since RAR gamma is predominantly expressed in adult skin, specific ligands for this subtype could (i) represent valuable tools to evaluate the biological role of RAR gamma in skin and (ii) provide therapeutic entities with a higher therapeutic index at lower teratogenic risk. Using in vitro binding studies and a functional transactivation assay, we have identified three compounds with high RAR gamma selectivity.

Animals↗
PubMed1992

Influence of formulation, receptor fluid, and occlusion, on in vitro drug release from topical dosage forms, using an automated flow-through diffusion cell.

An automated flow-through diffusion cell apparatus was used for comparing the release rates of a naphthoic acid derivative, CD 271, from different topical formulations. The influence of the following parameters on CD 271 release from the formulations was investigated: receptor fluid composition, occlusion, weight of tested formulation, and dosage form type. The amount of tested formulation was shown to have no significant effect on the apparent release constant and lag time for an anionic oil-in-water emulsion and an aqueous gel. Occlusion affected drug release from the different dosage forms. Thus, occlusion increased CD 271 pharmaceutical availability for a lotion and a hydroalcoholic gel containing 0.1% of solubilized drug. The release profile of CD 271 from the formulations was highly dependent on the receptor fluid composition. Drug release was dramatically enhanced with n-octanol as compared to an aqueous solution of surfactant. Using occlusive or nonocclusive procedures, CD 271 apparent release constant and lag time were found to be highly dependent on the type of tested formulation. The flow-through diffusion cell proposed in the present study allows an accurate comparison of drug release characteristics from prototype topical formulations and therefore represents a valuable tool for formulation research or quality control process.

Adapalene↗
PubMed1992

Expression of keratinocyte transglutamine mRNA revealed by in situ hybridization.

Plasma membrane-bound transglutaminase (TGm) catalyzes the formation of cornified envelopes (CE) in terminally differentiating keratinocytes. The recent cloning of cDNA encoding rabbit TGm allows detailed studies of its gene expression and regulation. In the present paper, we describe the localization of TGm mRNA in rabbit tissues, as well as in normal and psoriatic human skin, as assessed by in situ hybridization. Furthermore, we correlate TGm mRNA localization with the distribution of the TGm protein detected by immunohistochemistry with a specific monoclonal antibody. In rabbit epidermis, TGm mRNA was expressed in suprabasal cells. The TGm protein was detected in the upper stratum spinosum and stratum granulosum. In rabbit esophagus, TGm mRNA and protein were already expressed to a high level in the first suprabasal cell layer, and their expression decreased in the more differentiated cells. In normal human skin, a small amount of TGm mRNA, restricted to the stratum granulosum, was found, whereas psoriatic skin samples contained high amounts of TGm mRNA in the suprabasal layers with a decreasing gradient into the rete ridges, i.e., the involutions of the epidermis into the dermal compartment. The TGm protein was absent from the rete ridges and confined to several cell layers expressing high levels of mRNA. There was virtually no difference between uninvolved psoriatic and normal epidermis.

Animals↗
PubMed1992

Control of epidermal differentiation by a retinoid analogue unable to bind to cytosolic retinoic acid-binding proteins (CRABP).

The role played by cytosolic retinoic acid-binding proteins (CRABP) in the control of differentiation and morphogenesis by retinoids remains unclear, which contrasts with the presence of these binding proteins in tissues known to be targets for retinoic acid effects. Human epidermis represents a good system to address this question because 1) the effect of retinoids on keratinocyte differentiation is well documented; 2) epidermis contains CRABP, and the amount of these proteins is modulated both by keratinization and retinoids; 3) the architecture of epidermis obtained in vitro by growing adult human keratinocytes on a dermal substrate can be modulated by retinoids added to the culture medium in a dose-dependent manner; and 4) most markers of epidermal differentiation are also modulated by retinoids in a dose-dependent manner. In this study, we compared, in dose-response experiments, the biologic activities of retinoic acid and CD271, a substance unable to bind to CRABP, but able to bind to nuclear retinoic acid receptors (RAR). Our results show that retinoic acid and CD271 exert similar controls on epidermal morphogenesis and keratinocyte differentiation, as shown by the inhibition of the synthesis of suprabasal keratins, filaggrin, and transglutaminase. Therefore, we exclude a qualitative role for CRABP in the control exerted by retinoids on the differentiation and morphogenesis of cultured human keratinocytes. Instead of being involved in the pathway via which retinoids control epidermal gene expression, CRABP might regulate the amount of intracellular-active retinoic acid and thus control quantitatively the intensity of biologic effects.

Adapalene↗
PubMed1992

Differential expression of calgranulin A and B in various epithelial cell lines and reconstructed epidermis.

The monoclonal antibody F12, raised against epidermal cells from a psoriatic lesion, decorated antigens highly expressed in psoriatic epidermis and in cultured normal human keratinocytes. In normal human skin, F12 reacted only with follicular keratinocytes. Characterization of the immunoprecipitated antigens by two-dimensional gel electrophoresis revealed their identity with calgranulin A and B. A semiquantitative study with various established epithelial cell lines demonstrated that the expression of calgranulin A and B in hyperproliferative keratinocytes correlates with their potential to undergo terminal differentiation. In epidermis reconstructed in vitro, the antigen expression was stimulated by retinoids and suppressed under vitamin A starvation.

Antibodies, Monoclonal↗
PubMed1992

Selective synthetic ligands for human nuclear retinoic acid receptors.

From a series of naphthalene and benzoic acid derivatives we have identified synthetic retinoic acid analogues exhibiting high selectivity for the nuclear retinoic acid receptors RAR alpha (Am 580), RAR beta (CD 2019) and RAR gamma (CD 437) as well as ligands sharing high affinities for all RAR subtypes (CD 367). The compounds were evaluated in two complementary screening systems: (1) binding to nuclear proteins extracted from COS-7 cells after transfection with the appropriate expression vectors, and (2) induction of plasminogen activator in the embryonic mouse teratocarcinoma cell line F9. All compounds behaved as retinoic acid agonists in the F9 test.

Binding, Competitive↗
PubMed1992

Effect of drugs on the early and late phase UV erythema.

The inhibition of erythema by drugs applied topically after irradiation with 0.2-0.8 J of 313 nm has been studied in healthy volunteers. Indomethacin and piroxicam markedly inhibited the erythema at 6 to 24 h after irradiation but erythema reappeared at 45 h. The results reported suggest that the UVB response is biphasic with an early phase responsive to nonsteroid anti-inflammatory drugs and a late phase which is not responsive. No effect on the intensity of the erythema was seen with betamethasone chloroquine and cetirizine. Oral intake of aspirin before and/or after irradiation did not influence the UV response.

Adult↗
PubMed1991

Determination of retinoid activity by an enzyme-linked immunosorbent assay.

In normal human keratinocytes, retinoic acid suppresses the expression of the plasma membrane associated enzyme transglutaminase (TGm) at the pretranslational level. This finding led us to develop an enzyme-linked immunosorbent assay (ELISA) for the evaluation of the biological activity of retinoids, i.e., natural and synthetic derivatives of vitamin A. In this assay, keratinocytes are cultured in a 96-well cluster in the presence of different retinoid concentrations. The expression of TGm is then quantified, without any extraction or purification step, using a TGm-specific monoclonal antibody and a peroxidase-conjugated secondary antibody. The dose-response curves obtained show this ELISA to be a sensitive and reproducible assay to determine the potency of retinoids.

Animals↗
PubMed1991

Modulation of cellular cholesterol and its effect on cornified envelope formation in cultured human epidermal keratinocytes.

When cultured human epidermal keratinocytes (NHK) reach confluence they start to differentiate and an increase in the total cellular cholesterol content is observed. This increase parallels the appearance of a characteristic feature of terminal keratinocyte differentiation, the spontaneous formation of cornified envelopes (CE). Synthesis of CE is catalyzed by the plasma membrane-associated transglutaminase (TGm). Supplementation of the medium with inhibitors of cholesterologenesis suppressed increase in cholesterol levels and CE formation but did not interfere with TGm expression or TGm activity. Modulation of the plasma membrane cholesterol-phospholipid ratio of confluent NHK cultures using either pure phospholipid liposomes or liposomes enriched in cholesterol strongly affected spontaneous CE formation. Pure phospholipid liposomes completely inhibited CE formation, whereas cholesterol-enriched liposomes ensured envelope formation, even in the presence of inhibitors of cholesterol synthesis. From these results we conclude that in differentiating NHK an increase in the cellular cholesterol level is part of the differentiation program and is essential for the spontaneous CE formation.

Cell Aggregation↗
PubMed1991

The respecification of limb pattern by new synthetic retinoids and their interaction with cellular retinoic acid-binding protein.

We describe here experiments to examine the role of cellular retinoic-acid-binding protein (CRABP) during the induction of limb duplication in the chick limb bud and regenerating axolotl limb by retinoids. A newly synthesised class of retinoic acid analogues have been used because among them, some have been specifically designed with the property of binding to the retinoic acid receptors, but not to CRABP. We can thus test whether binding to CRABP is an obligatory step during limb respecification. The binding of four of these compounds to chick limb bud and axolotl CRABP was tested in sucrose density gradient assays and then their potencies at inducing limb duplications tested. Two of the four compounds do not bind to limb CRABP and yet are able to induce limb duplications, suggesting that an interaction with CRABP is not an obligatory step in the process. However, the two compounds which do bind to CRABP are more potent than the two which do not, suggesting that an interaction with CRABP may, nevertheless, increase the potency of a retinoid.

Ambystoma mexicanum↗
PubMed1991

Quantification of epidermal histological changes induced by topical retinoids and CD271 in the rhino mouse model using a standardized image analysis technique.

The rhino mouse has been used as an experimental model to screen topically active comedolytic agents. Adult rhino mice were treated on the back once daily for 5 consecutive days per week during 3 weeks. Skin histological preparations were analyzed by image analysis techniques to quantify the number of epidermal comedones, comedo profile and epidermal thickness. Using both a negative (treated with acetone) and a positive (treated with Aberel gel 0.025%) control group of animals in all experiments conducted over a period of about 3 years, we defined the upper and lower limit of acceptability of the results. Topical treatment with an acetone solution of all-trans retinoic acid (0.01, 0.03, 0.1%) and 13-cis-retinoic acid (0.1%) induced comedolysis and a marked increase in epidermal thickness. Commercial preparations of all-trans retinoic acid (Aberel lotion, gel and cream, Retin A cream, Retacnyl cream) presented a similar comedolytic activity. However, the epidermal thickening was higher with Retin A and weaker with Retacnyl. CD271, a new modulator of cell differentiation, applied either in acetone solution (0.01, 0.1%) or in lotion, gel or cream formulations (0.1%) also demonstrated a marked activity (i.e. comedolysis and epidermal thickening). These data confirm that the rhino mouse model can be used to assay drugs applied either in solvent or in topical formulations. Activity in this model compares favorably with published clinical observations in the treatment of acne.

Acne Vulgaris↗
PubMed1991

Selective high affinity retinoic acid receptor alpha or beta-gamma ligands.

Biological effects of retinoic acid (RA) are mediated through its binding to three closely related nuclear receptors (RAR alpha, RAR beta, and RAR gamma) belonging to the steroid-thyroid nuclear receptor family. RARs are able to modulate the transcription of specific genes by binding to responsive elements located in the promoter-enhancer region of these genes. As demonstrated by in situ hybridization, the distribution of each RAR type in the developing embryo, as well as in the adult, is not uniform. In this context, synthetic retinoids that would behave as selective ligands would be invaluable for studying the respective roles of each RAR type in cultured cells, whole animals, and embryos. Moreover, from a pharmacological point of view, such selective compounds may possess a higher therapeutic index and a lower teratogenic risk, because they might affect specific tissues and spare some others. As an approach to this problem, we have set up two complementary assays, (i) an in vitro binding assay to determine the Kd values of retinoids for RAR alpha, RAR beta, and RAR gamma and (ii) a functional assay in cultured cells to evaluate the potential of retinoids to transactivate, through their binding to one type of RAR, a reporter gene. The binding assay uses nuclear extracts of COS-7 cells transfected with vectors expressing RAR alpha, RAR beta, or RAR gamma. The functional assay is a measure of chloramphenicol acetyltransferase (CAT) activity in HeLa cells co-transfected with the expression vectors used in the binding assay and the reporter gene TRE-tk-CAT. Selective agonists for RAR alpha (Am80 and Am580) and RAR beta-RAR gamma (CD495 and CD564) were identified. However, compounds with pure RAR beta or RAR gamma selectivity have not yet been identified.

Carrier Proteins↗
PubMed1990

In vitro binding of retinoids to the nuclear retinoic acid receptor alpha.

We describe a rapid method for measuring in vitro binding properties of new synthetic retinoids to the recently identified nuclear receptor RAR alpha. Transfection of cos-7 cells with the expression vector RAR alpha O produces a 100-fold increase in intracellular RAR alpha concentration which allows us to perform accurate determination of binding parameters of various retinoids. Cytosol and nuclear extracts obtained after freeze drying of the transfected cells are incubated with a new stable tritiated analog of retinoic acid, [3H]CD367. Complete separation between RAR alpha and endogenous cellular retinoic acid binding protein is achieved by high-performance size-exclusion chromatography. These improved techniques provide a useful method for determining binding affinities of analogs to RAR alpha.

Animals↗
PubMed1990

The relevance of secondary radicals in the mode of action of anthralin.

The formation of free radicals during the reaction of anthralin analogues with peroxidizing polyunsaturated lipids was monitored by ESR spectroscopy. The biological effect of the different compounds was assessed by their ability to inhibit respiration of cultured human keratinocytes. C(10)-monosubstituted analogues of anthralin exhibited a strong antirespiratory effect and produced a cascade of radicals. Abstraction of the hydrogen atom at C(10) led to the generation of primary radicals which further decomposed into secondary radicals similar to those observed with anthralin itself. 10, 10'-disubstituted analogues of anthralin did not form any paramagnetic species during reaction with peroxidizing lipids while decomposition of a 2,7-disubstituted anthralin derivative under the same conditions resulted in primary, but not secondary radical species. Since both types of disubstituted analogues are devoid of antirespiratory activity we postulate that the antimitochondrial and thus antiproliferative activity of anthralin and its analogues is associated with their capacity to form secondary radicals during their decomposition.

Anthralin↗