Search PubMed⌕ Search

Biomedical subjects

B Shroot

Publications and source records attributed to B Shroot.

At least 37 records · Page 2Linked to original sources
PubMed1990

Reconstituted epidermis: a novel model for the study of drug metabolism in human epidermis.

The metabolic capacity of reconstituted epidermis from the outer root sheath cells of human hair follicles was determined. It was found that this epidermis possesses enzymes involved in both phase I (oxidation) and phase II (conjugation) reactions for drug biotransformation. The use of model substrates allowed the characterization of several isoenzymes. The homogenate fraction contained membrane-bound mixed-function oxydases (cytochrome P-450 dependent) involved in the O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzoxyresorufin, NADPH cytochrome c (P-450) reductase, testosterone 5 alpha-reductase, and UDP-glucuronosyltransferases, which conjugate 1-naphthol and bilirubin. One isoform of each glutathione S-transferase, steroid-, and arylsulfatases, acting on estrone- and 4-methylumbelliferone sulfates, was detected. Additionally, the activity of two distinct forms of epoxide hydrolases, which hydrate cis- and trans-stilbene oxides, could be measured. The presence of these drug metabolizing enzymes in the reconstituted epidermis indicates that it has a potential to serve as a model to study epidermal drug metabolism in vitro.

Arylsulfatases↗
PubMed1990

Reconstructed skin from cultured human keratinocytes and fibroblasts on a collagen-glycosaminoglycan biopolymer substrate.

A lattice prepared from biopolymer substrate bovine skin collagen and chondroitin-6-sulfate (glycosaminoglycan) served as a support for normal human keratinocytes and fibroblasts. Air exposure of the lattice on an agarose block gave rise to reconstructed epidermis, the histological features of which are very similar to normal human epidermis. Indirect immunofluorescence staining of the plasma membrane-associated transglutaminase, the enzyme responsible for the synthesis of the cornified envelope, revealed the same tissue distribution as observed in vivo. Cell cycle analysis showed a large shift of the normal human keratinocyte population into S-phase and cell division during the 1st week postinoculation. Furthermore, the effects of two modulators of differentiation (25-hydroxycholesterol and sodium butyrate) on the reconstruction of the epidermis were evaluated.

Animals↗
PubMed1990

Differentiation of F9 embryonal carcinoma cells by synthetic retinoids: amplitude of plasminogen activator production does not depend on retinoid potency or affinity for F9 nuclear retinoic acid receptors.

Retinoic acid and analogues (retinoids) are able to induce the differentiation of F9 murine embryonal carcinoma stem cells into endoderm-like cells. The secretion of plasminogen activator (PA) which accompanies this differentiation is a good index of the biological response of F9 cells to retinoids. We have previously reported that the potency of a series of natural and synthetic retinoids, evaluated by the concentration which provokes half-maximal induction of PA, correlates well with the affinity of these compounds for the endogenous F9 nuclear retinoic acid receptors, but not for the cytosolic retinoic acid binding protein, CRABP. In this paper we show that various retinoids differ, not only in terms of potency, i.e. the dilution at which they are active, but also in terms of the amount of PA that they induce. This parameter, called amplitude, is used to quantify the extent of PA induction by a given retinoid relative to retinoic acid. The amplitude parameters of synthetic retinoids are found to vary over a wide range and are independent of both potency and binding affinity for F9 retinoic acid receptors. It is proposed that the amplitude of the biological response to a given retinoid is the resultant of three factors: (i) the total or partial agonist character of the retinoid; (ii) the binding spectrum of the retinoid for the various types of retinoic acid receptors; (iii) the chemical and metabolic stability of the retinoid in the test system.

Animals↗
PubMed1990

The physiology and biochemistry of retinoic acid.

The ability of the fat soluble Vitamin A to modulate cellular differentiation has been known for over 60 years. Numerous studies have shown that Vitamin A and its 2 major metabolites play a key role in vision (retinal) and cell differentiation (retinoic acid, RA). The control of cellular differentiation may be exercised on a variety of tissues including simple and stratified epithelia, the latter will be the focal point of this article, with emphasis being given to the skin. In the development of the limb bud, RA has been proposed as a morphogen controlling development via a concentration gradient established by a mechanism thought to involve specific nuclear receptors and a cytosolic binding protein (CRABP). In the hemopoietic system, RA is known to modulate several functions of polymorphonuclear leucocytes (PMN) in vitro and the differentiation of granulocyte macrophage-precursors. Moreover it has been shown to exert immunomodulatory properties.

Adjuvants, Immunologic↗
PubMed1989

Retinoic acid controls expression of epidermal transglutaminase at the pre-translational level.

Human epidermal keratinocytes were cultured until sub-confluence in low Ca2+ (0.15 mM) serum-free synthetic MCDB 153 medium. Raising the Ca2+ concentration to 1.15 mM caused an increase in envelope competence as well as plasma membrane associated transglutaminase (TGm) activity. This increase was not observed when the high Ca2+ medium contained retinoic acid. Immunofluorescence studies as well as immunoblotting with the TGm-specific monoclonal antibody B.C1 revealed that retinoic acid inhibits expression of TGm. Isolation and in vitro translation of mRNA with subsequent immunoprecipitation showed that retinoic acid inhibits TGm expression at the pretranslational level.

Antibodies, Monoclonal↗
PubMed1989

Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes.

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross-linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus-40 (SV-40)-transformed human foreskin keratinocyte line SV-K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane-associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane-associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.

Butyrates↗
PubMed1989

Bioassays for retinoic acid-like substances using cultured human keratinocytes.

Using cultured human keratinocytes, three bioassays for retinoic acid-like substances have been developed. They are based on the ability of these substances (1) to inhibit cross-linked envelope formation, (2) to inhibit the synthesis of the K1 keratin, and (3) to stimulate the synthesis of the K19 keratin. The data, expressed as 50% inhibitory or activating concentrations were used to rank compounds according to their activity.

Autoradiography↗
PubMed1988

Plasma membrane transglutaminase and cytosolic transglutaminase form distinct envelope-like structures in transformed human keratinocytes.

Cross-linked envelope formation in the transformed human keratinocyte line SV-K14 requires treatment of the cells with a Ca2+ ionophore. Depending on the culture conditions, different extracellular Ca2+ concentrations are necessary to trigger the process which is catalyzed by the enzyme transglutaminase. Confluent cells grown in the presence of serum express only the cytosoluble form of the enzyme and need 5 mM Ca2+ for optimum protein cross-linking, whereas serum-starved cells which additionally contain the plasma membrane associated form of the enzyme require only 1 mM Ca2+. The envelope-like structures thus synthesized are morphologically and biochemically distinct.

Cell Line↗
PubMed1988

An improved assay procedure and a new chemically stable ligand for cytosolic retinoic acid binding protein.

A high through-put method to assay for binding of retinoids to their cytosolic binding protein is described. The protein-bound retinoid is quantified following its complete separation from the free ligand by a single gel filtration chromatography step on Sephadex G-25 columns. The method allows a single person to process up to 100 samples per day. A new, substituted benzo[b]thiophene carboxylic acid derivative (tritiated) is proposed as an alternative ligand for cytosolic retinoic acid binding protein. This new analog (CD270), which contains no olefinic double bonds, is characterized by its chemical stability to light and atmospheric oxidation. This molecule shows binding characteristics similar to those of retinoic acid, both in terms of binding affinity and binding specificity (Kd of about 2 nM), and offers the additional advantage that the unbound molecule is absorbed on Sephadex G-25, while the protein-bound ligand is unaffected by the carbohydrate gel matrix. When the affinities of retinoic acid and several analogs were determined by competition binding experiments, similar results were obtained when either tritiated retinoic acid or tritiated CD270 were used as the labeled ligand. The tritiated, heterocyclic retinoic acid analog, CD270, is thus proposed as an alternative ligand for cytosolic retinoic acid binding protein.

Animals↗
PubMed1988

Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro.

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.

Blotting, Western↗
PubMed1988

Transglutaminases in normal and transformed human keratinocytes in culture.

The transglutaminases of cultured normal and transformed human keratinocytes (line SV-K14) are characterized. Both cell types display two forms of the enzyme, one of which is cytosoluble (TGc) and the other which is associated with the plasma membrane (TGm). Normal keratinocytes contain predominantly TGm, and SV-K14 cells mainly TGc. The ratio of TGm to TGc can be modulated by the culture conditions and correlates with the competence of the cells to form a cornified envelope. TGm and TGc differ in their biochemical and immunological properties. SDS electrophoresis reveals apparent molecular weights of 92 and 85 kD, respectively. Only the activity of TGc is inhibited in the presence of guanosine 5'-triphosphate. Their response to Ca2+ is different: TGc exhibits a sigmoidal activation kinetics with an A50 value of about 200 microM, whereas the kinetics for TGm is hyperbolic with an A50 value of 75 microM. TGm reacts with a monoclonal antibody raised against epidermal "particulate" transglutaminase, and TGc with a polyclonal antibody raised against guinea pig liver transglutaminase. These reactions are very specific and no cross-reaction occurs. The coappearance of TGm with a proteolytic fragment (Mr 82,000) in the cytosol and intracellular particulate fraction of normal human keratinocytes is probably a preparation artifact.

Cell Line, Transformed↗
PubMed1988

Morphological and biochemical characterization of the cornified envelopes from human epidermal keratinocytes of different origin.

The formation of a cornified envelope (CE) is a major event in the terminal differentiation of epidermal cells. Nomarski contrast microscopy of the envelopes purified from different sources reveals the existence of two major, but morphologically distinct classes: the very irregularily shaped fragile type CEf, and the polygonal rigid type CEr. Human keratinocytes in submerged culture are only able to produce type CEf. Specimens from healthy human epidermis contain largely type CEr. Psoriatic scales from different patients show both types in varying proportions. Tape stripping of normal epidermis reveals that type CEf is present in the lowermost layers of the stratum corneum and type CEr is present in the upper layers, indicating that the two types represent a different stage of maturation. Cyanogen bromide peptide mapping of electrophoretically purified envelopes reveals striking differences between cultured keratinocytes, normal epidermis, and psoriatic scales but also slight interindividual variations. This variability supports the view that the molecular CE composition is not strictly determined. On the other hand, no difference could be detected in the peptide maps of CEf and CEr obtained after tape stripping from the same healthy volunteer indicating that CE maturation within the stratum corneum does not involve the provision of qualitatively new proteins.

Cell Differentiation↗
PubMed1988

Hexadecane-induced skin hyperplasia in the hairless rat: time course of histological and biochemical events related to the synthesis of polyamines and DNA.

Several biochemical parameters including ornithine decarboxylase activity (ODC) and tissue polyamine levels were measured during the hexadecane-induced epidermal hyperplasia of hairless rat skin. Animals received three applications of 200 microliters pure n-hexadecane on day 1. ODC activity and polyamine levels (putrescine, spermidine and spermine) in the epidermis were significantly increased and reached maximum elevations at 12 h after the start of n-hexadecane treatment with DNA synthesis peaking at 24 h. Histological studies confirmed a significant cellular edema at 24 h after the beginning of the treatment followed at 48 h by an epidermal hyperplasia which was maximum at 72 h. These data support the view that ODC activation, increased biosynthesis of polyamines and DNA are early events in epidermal cell hyperproliferation.

Alkanes↗
PubMed1988

Role of free radicals in the mode of action of anthralin.

Radical reactions of anthralin and its metabolites with skin have been studied by ESR spectroscopy. The influence of compounds which are known to suppress inflammation are described. The ESR spectra recorded during the reaction of anthralin with skin were essentially composed of one broad line centered at g = 2.0030. Similar but much weaker spectra were recorded with the dimer and no signal at all was obtained with 9,10-dihydroxyanthraquinone. The ESR response obtained with anthralin was neither affected by the radical scavengers 2-tert-butyl-4-methoxy-phenol (BHA), 2,6-di-tert-butyl-4-methyl-phenol (BHT) dl-alpha-tocopherol, nor by the antiinflammatory agents clobetasol-17-propionate or indomethacin, nor by potassium hydroxide. We infer that anthralin inflammation is not associated with the presence of anthralin-derived radicals in the skin.

Animals↗
PubMed1988

Use of the chick embryo for pharmacological screening of retinoids.

Retinoic acid and some selected analogs were tested to evaluate their effect on skin morphogenesis and toxicity in the chick embryo. Retinoids dissolved in dimethyl sulfoxide were injected at doses varying from 10 pmol to 10 mumol into the amniotic cavity of 10-day-old chick embryos (n = 20). At 16 days of incubation, the eggs were opened to record the number of dead embryos and the number of embryos presenting club-shaped feathers. A lethal embryotoxic dose (LED50d16) which, at 16 days of incubation, provokes the death of 50% of the embryos and an effective dose (ED50) which induces production of club-shaped feathers in 50% of surviving embryos were then calculated using log-probit analysis. Retinoids could be classified according to their ED50. For example, arotinoid Ro 13-7410 appears approximately 1,000 times more active than all transretinoic acid. However, the analogs which are more active are also more toxic. The assay described in the present study appears to be a simple and useful model for the screening of retinoids.

Animals↗
PubMed1988

Biological activity of retinoids correlates with affinity for nuclear receptors but not for cytosolic binding protein.

In order to better understand the respective roles of the nuclear retinoic acid receptors (RARs) and the cytosolic retinoic acid binding protein (CRABP) in the mode of action of retinoic acid (RA), several types of RA analogs have been synthesized. Representative compounds have been radiolabeled to a high specific activity and their binding (direct and competition) to RARs and CRABP was determined. Their biological activity on F9 embryonal carcinoma cell differentiation has been determined by a quantitative assay of plasminogen activator (PA). All biologically active analogs studied in this work bound to RARs. A good correlation was found between PA induction and affinity for the RARs, with the exception of RA itself which was a good ligand but a moderate inducer of F9 differentiation. Two biologically active analogs (compounds II and III) did not bind to the CRABP. One biologically inactive analog (compound VIII) bound to CRABP. These results strongly suggest that retinoids must bind to RARs but not necessarily to CRABP in order to induce cell differentiation in F9 cells.

Animals↗