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Biomedical subjects

G Siegenthaler

Publications and source records attributed to G Siegenthaler.

At least 37 records · Page 2Linked to original sources
PubMed1995

Amino acid sequence analysis of human S100A7 (psoriasin) by tandem mass spectrometry.

The Ca(2+)-binding proteins regulate a number of cellular and extracellular activities and deregulations of S100 gene expression are associated with several human diseases. For example, S100A7 is upregulated in psoriatic skin, implicating a link with psoriasis, a chronic inflammatory dermatosis. We purified human S100A7 and determined its protein sequence by tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A7 with all known human S100 proteins showed that S100A7 is the most divergent of all S100 proteins.

Amino Acid Sequence↗
PubMed1995

Characterization of two post-translationally processed forms of human serum retinol-binding protein: altered ratios in chronic renal failure.

Retinol-binding protein (RBP) is the specific blood carrier for the transport of retinol (vitamin A) to target tissues. As the kidney is involved in RBP metabolism, the analysis of RBP species in the serum of patients with chronic renal failure (CRF) was used as a model to study possible RBP alterations. SDS-PAGE-immunoblotting analysis of normal and CRF sera shows a doublet of RBP bands (band A and band B) near 21 kDa. Mass spectrometric analysis of purified RBPs from CRF and normal sera revealed the presence not only of full-length RBP (183 residues, migrating in band A) but also two forms of RBP differing from the native form by the loss of C-terminal Leu (i.e., RBP1 (residues 1-182), migrating in band A also) and the loss of C-terminal Leu-Leu (i.e., RBP2 (residues 1-181), migrating in band B). Interestingly, RBP2 was considerably increased in the serum of CRF, whereas it was low in normal sera. In healthy retinol target-tissues and in cultured HepG2 cells, RBP2 levels were significantly and variably present compared to RBP and RBP1. We propose that these post-translationally modified forms of RBP occur in cells and that after their release into the blood circulation RBP2 is cleared by the kidney in healthy individuals but accumulates in the serum of CRF patients. RBP2 may have an important physiological role in retinol transport and/or recycling.

Amino Acid Sequence↗
PubMed1994

Differential expression of cyclophilin isoforms during keratinocyte differentiation.

Cyclophilin A, the major intracellular binding protein for the immunosuppressive drug cyclosporin A (CsA), was studied in human keratinocytes during differentiation both in vivo and in vitro. Analysis of cyclophilin by gel-filtration radiobinding-assay with tritiated CsA showed one specific radioactive peak at 17 kDa. By this technique, the levels of cyclophilin (mean 55.23 +/- 8.43 pmol/mg protein) did not significantly differ during keratinocyte differentiation. When the protein extracts from calcium-induced differentiating keratinocytes and normal human skin were analysed by PAGE radiobinding-assay, two specific radioactive CsA-binding peaks were detected. The major peak (RF 0.13) was expressed in all samples (mean 47.32 +/- 17.53 pmol/mg protein) whereas the minor peak (RF 0.23) was dramatically decreased about 6-fold in abnormally differentiated skin (psoriasis) as well as in non-differentiated keratinocytes. At least six [3H]CsA-binding isoforms with pI values ranging from 5.58 to 7.75 were detected by isoelectrofocusing autoradio-blotting-assay in normal human skin; three of them immunoreacted with antibodies to cyclophilin. These results demonstrated the presence of several cyclophilin isoforms in human epidermal cells and an expression which correlated with the differentiation of human keratinocytes both in vivo and in vitro.

Amino Acid Isomerases↗
PubMed1994

Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro.

Epidermal fatty acid-binding protein (E-FABP) was isolated from human skin and purified to homogeneity. Its molecular mass was estimated to be 15 kDa and the pI of non-denaturing protein was 5.6. Scatchard-plot analysis revealed one class of binding site for oleic acid with a Kd of 0.46 microM. Structure-binding relation experiments revealed a high affinity of E-FABP for stearic acid which decreased on reduction of the number of carbon atoms or introduction of double bonds into the fatty acid chain. Squalene, cholesterol and retinoic acid isomers showed no affinity, suggesting that E-FABP displays high specificity for fatty acids. E-FABP is a scarce cytosolic protein (0.065% of total protein). Only trace amounts could be detected in normal human skin but up to 42.5 +/- 3.4 pmol/mg of protein was found in a non-malignant defect of keratinocyte differentiation (psoriatic lesions). E-FABP levels were low in cultured human keratinocytes grown under proliferation-stimulating conditions but increased about 2-fold on induction of differentiation by Ca2+. Immunohistochemical localization showed cytosolic staining in differentiated cells of normal and psoriatic skin, suggesting a link between E-FABP and keratinocyte differentiation. The presence of E-FABP in tissues other than skin (heart, intestine and adipose tissue) excludes its specific role in fatty acid metabolism in epithelial cells or its involvement in skin lipid-barrier function.

Amino Acid Sequence↗
PubMed1994

Topical retinaldehyde on human skin: biologic effects and tolerance.

The present study was designed to explore if *etinaldehyde, a natural metabolite of vitamin A, has any biologic activity and is tolerated by human skin. Biologic activity was shown by the induction of cellular retinoic acid-binding protein type 2 (CRABP 2) mRNA and protein; the rank order for CRABP-2 increase was retinoic acid > retinaldehyde > 9 cis retinoic acid > retinol > beta carotene. In volunteers treated 1-3 months with 0.5, 0.1, and 0.05% retinaldehyde, there was a dose-dependent and significant increase in epidermal thickness, keratin 14 immunoreactivity, and Ki67-positive cells. The area of distribution of involucrin, transglutaminase, and filaggrin immunoreactivity was also increased in a dose-dependent manner, and keratin 4 immunoreactivity was induced in the upper epidermis. In pilot clinical tolerance studies, 229 patients received topical retinaldehyde at different concentrations; the 1% preparation was tolerated by up to 70% of the treated subjects; tolerance of the 0.5% preparation was slightly better, whereas both 0.1 and 0.05% preparations applied on facial skin were well tolerated and allowed prolonged use (up to 3 years) in patients with inflammatory dermatoses. These findings indicate that topical retinaldehyde has biologic activity and is well tolerated on human skin.

Administration, Topical↗
PubMed1994

Topical glucocorticosteroids modulate the expression of CRABP I and II in human skin differently.

Epidermal cells express two retinotic acid-binding proteins (CRABP I and II). Because CRABP II protein is strongly induced by topical retinoic acid, the respective roles of the two proteins in the pharmacological activity and toxicity of topical retinoids deserve particular attention. Since topical steroids diminish the irritation induced by retinoic acid (RA), whereas retinoic acid may counteract the atrophogenic effects of steroids, the possible interplay of both compounds in the expression of CRABP I and II appeared worth studying. We have analyzed the effects of topical application of triamcinolone acetonide (TA) on the retinoic acid-induced altered expression of CRABP I and II in normal human skin, at the protein and mRNA levels. We found that CRABP II protein and mRNA were strongly increased upon retinoic acid application: this induction was significantly inhibited by concomitant application of triamcinolone acetonide; a more potent steroid, difluocortolone valerate, was also found to diminish normal endogenous expression of CRABP II. In contrast, CRABP I protein was decreased by topical retinoic acid, and the down modulating effect of retinoic acid was counteracted by triamcinolone acetonide.

Administration, Topical↗
PubMed1993

9-cis-retinoic acid is a natural antagonist for the retinoic acid receptor response pathway.

The pleiotropic activities of retinoids are mediated by two types of nuclear receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). All-trans-retinoic acid (RA) transcriptionally activates RARs, but not RXRs, whereas its natural stereoisomer, 9-cis-RA, is the ligand for RXRs. Here, we demonstrate that 9-cis-RA did not transcriptionally activate RARs, whereas in the presence of all-trans-RA the transactivation of RARs was inhibited in a dose-dependent manner by 9-cis-RA. RAR homodimer complexes were destabilized in vitro in the presence of 9-cis-RA. This suggests that 9-cis-RA may be a natural antagonist of all-trans-RA for binding to RAR complexes. The levels of 9-cis-RA may determine by which pathway the transcription of retinoid-responsive genes is modulated.

Animals↗
PubMed1993

Characterization and expression of a novel human fatty acid-binding protein: the epidermal type (E-FABP).

Using PAGE--Autoradioblotting technique we have characterized an E--FABP in human epidermal cells that is distinct from liver-, heart-, intestine- and adipose tissue-FABPs. FABP radiobinding analysis was performed directly on protein extracts without prior partial purification. E-FABP has a Mr of approximately 15 kDa and binds oleic acid with high affinity but does not bind all-trans-, 13-cis- and 9-cis-retinoic acid nor all-trans-retinol. Expression levels of E-FABP were low in normal epidermis, higher in human cultured keratinocytes and still higher in psoriasis, a disease characterized by abnormal epidermal differentiation. These findings suggest that epidermal cells may have a distinct fatty acid metabolism compared to other tissues.

Carrier Proteins↗
PubMed1992

Expression of CRABP-I and -II in human epidermal cells. Alteration of relative protein amounts is linked to the state of differentiation.

The physiological role of cellular retinoic acid-binding proteins (CRABPs) may be to influence the intracellular level of free retinoic acid in the cell. In the present study two isoforms of CRABP, CRABP-I and CRABP-II were partially characterized in various human Malpighian epithelia and in human cultured keratinocytes expressing various patterns of differentiation. We have developed a new sensitive radiobinding assay using a PAGE/autoradioblotting technique which effectively separates CRABP-I and CRABP-II. This method allows the simultaneous quantification of these proteins. We show that CRABP-I and -II have similar M(r) values (15,000), but differ in their dissociation constant towards retinoic acid (Kd of 16.6 nM and 50 nM respectively), in pI (4.86 and 5.13) and in their relative mobilities (RF) on PAGE under nondenaturating conditions (RF values 0.65 and 0.44). In addition, we show that CRABP-II is the major isoform expressed in human keratinocytes, in vivo as in vitro. Furthermore, we demonstrate that CRABP-II is actually the CRABP previously studied in epidermal cells by a PAGE assay (Siegenthaler & Saurat (1987) Eur. J Biochem. 166, 209-214) and whose levels are dramatically increased by retinoic acid and its analogues in human epidermis. Keratinocytes, in the absence of full terminal differentiation, as well as hyperplasia, such as cultured human differentiating keratinocytes, psoriatic plaques, and non-keratinized oral mucosa, contained high levels of CRABP-II. CRABP-I was not detected in cultured keratinocytes, whereas normal skin (at full terminal differentiation) expressed CRABP-I and CRABP-II at a ratio of approx. 1:1.4. This value was approx. 1:17 in lesional psoriatic skin and 1:8 in oral mucosa. These observations suggest that CRABP-I and -II are regulated differently in human keratinocytes. The sharp increases in CRABP-II levels are associated with an alteration in the differentiation programme, as well as with cell response to retinoic acid overload, whereas CRABP-I might be a marker for terminal differentiation.

Animals↗
PubMed1992

Domains of cellular retinoic acid-binding protein I (CRABP I) expression in the hindbrain and neural crest of the mouse embryo.

We describe here the distribution of cellular retinoic acid-binding protein I (CRABP I) in the head of the early mouse embryo from day 8 to day 13 of gestation, using both in situ hybridisation to localise mRNA and immunocytochemistry to localise protein. The distribution of mRNA and protein was found to be identical. CRABP I first appeared in part of the presumptive hindbrain of the presomite embryo and then became localised to rhombomeres 2, 4, 5 and 6. The only other area of expression in the cephalic neuroepithelium was in a part of the midbrain roof. The neural crest and its mesenchymal derivatives, the branchial arches, expressed CRABP I and crest could be seen streaming from the neuroepithelium of individual rhombomeres into particular branchial arches. This suggested a fate map could be constructed describing the rhombomeric origin of branchial arch mesenchyme. Later in development, axons throughout the hindbrain expressed CRABP I. The results are considered in terms of the role of retinoic acid in the specification of neuronal phenotype in the hindbrain and in axon outgrowth.

Amino Acid Sequence↗
PubMed1992

Expression of cellular retinoid-binding proteins during normal and abnormal epidermal differentiation.

Retinoids have important roles in growth and differentiation of epidermal cells. We have analyzed the expression of two intracellular retinoid-binding proteins, the cellular retinol-binding protein type I and the cellular retinoic acid-binding protein type I, during normal and abnormal epidermal differentiation. Both proteins were found to be expressed in normal epidermis with increasing expression from basal layer towards superficial layers. In psoriatic lesions, a hyperproliferative condition of the skin, the epidermal expression of cellular retinol-binding protein I was induced, whereas expression of cellular retinoic acid-binding protein I was sharply down-regulated. This and other features of psoriatic lesions indicate that down-regulation of cellular retinoic acid-binding protein I expression might cause aberrant retinoid-regulated gene expression in skin. In basal and squamous cell carcinomas, cellular retinoic acid-binding protein I expression was down-regulated, whereas cellular retinol-binding protein I was expressed. Apart from epidermal cells, a mesenchymal, dendritic cell-type, strongly expressing cellular retinoic acid-binding protein I, was identified in the dermis. In several hyperproliferative conditions of the skin, including psoriasis, and squamous and basal cell carcinomas, this cell type was abundant. These results have implications for the role of retinoids in normal and abnormal epidermal differentiation and suggest that part of the phenotype of psoriasis is due to inappropriate metabolism of retinoic acid in skin.

Adult↗
PubMed1992

Overexpression of cellular retinoic acid-binding protein type II (CRABP-II) and down-regulation of CRABP-I in psoriatic skin.

Cellular retinoic acid-binding proteins (CRABPs) might exert a physiological function by controlling the intracellular levels of free retinoic acid. The aim of this study was to analyze the relative expression of CRABP-I and CRABP-II in lesional (LPS) and nonlesional (NLPS) psoriatic skin. CRABP-I and -II proteins were analyzed by a PAGE-autoradioblotting technique, and their respective mRNA were studied by RNA blot and in situ hybridization. We found that CRABP-II levels were 6-fold higher in LPS and 2-fold in NLPS as compared to normal skin, whereas CRABP-I levels were decreased in NLPS and LPS. CRABP-II mRNA were grossly overexpressed in all LPS and some NLPS specimens. These results indicate a switch to the overexpression of CRABP-II mRNA in psoriasis which induces high levels of the protein mainly in LPS; these observations may be relevant to the pathophysiology and therapy of psoriasis as CRABP-I and -II have different ligand-binding affinities.

Carrier Proteins↗
PubMed1991

Binding of synthetic analogues of retinol and retinoic acid (CD-270 derivatives) to retinoid-binding proteins.

Cellular retinoid-binding proteins play an important physiological role in the mode of action of retinoids upstream from the nuclear receptors. Tritiated analogues of retinol (ROL) and retinoic acid corresponding to substituted benzo[b]thiophene (CD-270) alcohol and carboxylic acid, respectively, were used for the binding studies of the cellular retinoic acid-(CRABP-) and retinol-(CRBP-) binding proteins in human epidermal cells and serum retinol-binding protein (RBP). We show that [3H]CD-270 carboxylic acid binds specifically to CRABP, whereas the alcohol derivative binds strongly to CRBP. However, the low amount of CRABP present concomitantly with CRBP suggests that [3H]CD-270 alcohol might be oxidized into minute amounts of the corresponding carboxylic acid derivative. The acidic character of the alcohol goup due to the aromatic ring of CD-270 might also support a possible binding to CRABP. In contrast, both derivatives show no affinity to RBP. These results suggest that the binding site of RBP is more restrictive to the retinoyl moiety than the binding site of cellular retinoid-binding proteins and therefore tolerates less chemical modification on the hydrophobic part of the ROL molecule. Moreover, when using these derivatives as ligands 2.4 times more CRABP and 3.9 times more CRBP could be measured with the PAGE technique as compared when using the natural [3H]ligands. The CD-270 derivatives might therefore be used as stable ligands for the study of both CRBP and CRABP. Since CD-270(OH) binds to the CRBP this may lead to the development of new synthetic analogues of ROL which could be used as tools for the study of the role of CRBP in the transport and metabolism of ROL.

Binding, Competitive↗
PubMed1990

Ligand specificities of recombinant retinoic acid receptors RAR alpha and RAR beta.

Binding of retinoic acid (RA) to specific RA receptors alpha and beta (RAR alpha and RAR beta) was studied. Receptors were obtained in two ways: (1) full-length receptors were produced by transient expression of the respective human cDNAs in COS 1 cells; and (2) the ligand-binding domains of RAR alpha and RAR beta were produced in Escherichia coli. RA binding to the wild-type and truncated forms of the receptor was identical for both RAR alpha and RAR beta, indicating that the ligand-binding domains have retained the binding characteristics of the intact receptors. Furthermore, RA bound with the same affinity to both RAR alpha and RAR beta. Only retinoid analogues with an acidic end-group were able to actively bind to both receptors. On measuring the binding of various retinoids, we have found that the properties of the ligand-binding sites of RAR alpha and RAR beta were rather similar. Two retinoid analogues were capable of binding preferentially to either RAR alpha or RAR beta, suggesting that it may be possible to synthesize specific ligands for RAR alpha and RAR beta.

Animals↗
PubMed1990

Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes.

Cultured keratinocytes offer an attractive model for studying the metabolism of retinol in relation to cell differentiation, since the extent of keratinocyte differentiation can be modulated experimentally. The metabolism of retinol and retinal was studied in cytosol fractions prepared from two distinct keratinocyte populations, differentiating and non-differentiated. The enzymic activities were analysed using physiological concentrations of [3H]retinol and [3H]retinal in the presence of cofactors. The products formed were quantified by h.p.l.c. In the population of differentiating keratinocytes, the formation of retinoic acid from retinol occurred at a rate of 4.49 +/- 0.17 pmol/h per mg of protein, but no such conversion was observed in the population of non-differentiated cells. However, when retinal was used as substrate, retinoic acid was formed in both cell populations, at rates of 14.4 pmol/h per mg of protein in non-differentiated and 51.6 pmol/h per mg of protein in differentiating keratinocytes. Using PAGE/radiobinding assay, we demonstrated that retinoic acid formed from retinol was bound in differentiating keratinocytes to endogenous cellular retinoic acid-binding protein (CRABP). Furthermore, retinal was reduced to retinol in the presence of NADH in both differentiating and non-differentiated keratinocytes at a similar rate (8 pmol/h per mg of protein). Although retinal could not be detected under physiological conditions, it was found in significant amounts at pH 8.5-9, which is optimal for enzymic activity. This indicates that in keratinocytes retinal is an intermediate metabolite in retinoic acid formation from retinol. The enzymes catalysing the conversion of retinol into retinoic acid were found to differ from other alcohol and aldehyde dehydrogenases, since the formation of retinoic acid was not significantly affected by specific inhibitors of alcohol metabolism, such as 4-methylpyrazole and disulfiram. Moreover, the cytosol of non-differentiated keratinocytes did not generate retinoic acid from retinol despite showing alcohol dehydrogenase activity. The results suggest that: (1) retinol metabolism in human keratinocytes is different from that of other alcohols, (2) retinal is an intermediate metabolite in the conversion of retinol into retinoic acid, and (3) differentiating keratinocytes rich in CRABP are probably target cells for retinoic acid action.

Adolescent↗