Penetration and biological effects of topically applied cyclosporin A nanoparticles in a human skin organ culture inflammatory model.

Systemic antipsoriatic therapies have potentially life-threatening, long-term side effects. The efficacy of topical drugs is poor, but may be improved by the use of delivery systems based on drug nanoparticles. To produce nanoparticles (NP) composed of cyclosporin A, a classical antipsoriatic drug, and to investigate their penetration and biological effects in human skin affected by psoriatic symptoms, poly-ε-caprolactone (PCL) and cyclosporin A (CsA) NP were prepared by the solvent evaporation method.

Evaluation of topically applied copper(II) oxide nanoparticle cytotoxicity in human skin organ culture.

The increasing use of nano-sized materials in our environment, and in many consumer products, dictates new safety concerns. In particular, adequate experimental models are needed to evaluate skin toxicity of metal oxide ions, commonly found in cosmetic and dermatologic preparations. We have addressed the biological effects of topically applied copper oxide (CuO) nanoparticles in human skin organ cultures, using light and electron microscopy, and biochemical tests.

Skin organ culture as a model to study oxidative stress, inflammation and structural alterations associated with UVB-induced photodamage.

Background: Ultraviolet (UV) irradiation is a major cause of skin damage, of long-term alteration of skin metabolism, homoeostasis and physical structure. The analysis of UV-induced pathogenic processes requires in vitro models allowing biochemical studies, and appropriate for the development of novel, accurate diagnosis methods based on non-invasive procedures.

Objectives: This work was aimed to reproduce the effects of UVB on whole-skin explants ex vivo and to study underlying biochemical mechanisms, especially in correlation with skin autofluorescence.

Strontium hexaferrite nanomagnets suspended in a cosmetic preparation: a convenient tool to evaluate the biological effects of surface magnetism on human skin.

Background/purpose: Magnetic therapy has been popular for ages, but its therapeutic abilities remain to be demonstrated. We aimed to develop a homogeneous, stable dispersion of magnetic nanoparticles in a skin-care preparation, as a tool to analyze the biological and physiological effects of superficial magnetism in skin.

Methods: SrFe(12)O(19) nanoparticles were generated by ultrasound, dispersed in glycerol, stabilized in Dermud cream and permanently magnetized.

Protective effects of a cream containing Dead Sea minerals against UVB-induced stress in human skin.

Background: Dead Sea (DS) mud and water are known for their unique composition of minerals, and for their therapeutic properties on psoriasis and other inflammatory skin diseases. Their mode of action, however, remains poorly known.

Objectives: To analyse the ability of Dermud, a leave-on skin preparation containing DS mud and other ingredients like DS water, zinc oxide, aloe-vera extract, pro-vitamin B5 and vitamin E, to antagonize biological effects induced by UVB irradiation in skin when topically applied in organ cultures.

Aged keratinocyte phenotyping: morphology, biochemical markers and effects of Dead Sea minerals.

The aging process and its characterization in keratinocytes have not been studied in depth until now. We have assessed the cellular and molecular characteristics of aged epidermal keratinocytes in monolayer cultures and in skin by measuring their morphological, fluorometric and biochemical properties. Light and electron microscopy, as well as flow cytometry, revealed increase in cell size, changes in cell shape, alterations in mitochondrial structure and cytoplasmic content with aging.

The ubiquitin-proteasome system at the crossroads of stress-response and ageing pathways: a handle for skin care?

The regulation of gene expression at the transcriptional level has been considered for long as the main mechanism of cellular adaptive responses. Since the turn of the century, however, it is becoming clear that higher organisms developed a complex, sensitive and maybe equally important network of regulatory pathways, relying largely on protein interactions, post-translational modifications and proteolysis. Here we review the involvement of the ubiquitin-proteasome pathway of protein degradation at different levels of cellular life in relation with ageing, and with a special focus on skin.

The interaction of pemphigus autoimmunoglobulins with epidermal cells: activation of the fas apoptotic pathway and the use of caspase activity for pathogenicity tests of pemphigus patients.

Pemphigus is a fatal autoimmune disease in which autoimmunoglobulins PV-IgG (binding to desmoglein 3) and PF-IgG (binding to desmoglein 1) in pemphigus vulgaris and pemphigus foliaceus, respectively, cause intraepidermal blisters, cell-cell separation (acantholysis), and cell death. The mechanism of acantholytic lesion formation has not yet been elucidated. Recently, we have reported that an apoptotic mechanism might be operative in PV-IgG-induced acantholysis: (1) in patients' lesional and some perilesional skin portions, the FasR pathway is activated as its components were enriched; (2) in cultured keratinocytes, PV-IgG upregulates effectors of the FasR pathway (including the mitochondrial loop), as found by immunodetermination (cytochemistry, Western blot of pathway effectors) and determination of caspases 1, 3, and 8 activity/activation; (3) in organ cultures of skin incubated with PV-IgG, activated caspase 8 was found also in perilesional cells and coaggregated with bound PV-IgG; (4) caspase 8 activation in DISCs precedes caspase 3 activation in keratinocytes in cultures upon incubation with PV-IgG.

Replicative senescence enhances apoptosis induced by pemphigus autoimmune antibodies in human keratinocytes.

We have recently shown that skin lesions of the autoimmune disease pemphigus vulgaris are associated with Fas-mediated apoptosis. Here, we describe the induction of the Fas-dependent apoptosis pathway in cultured keratinocytes by pemphigus vulgaris autoantibodies (PV-IgG), as seen from a variety of cellular, morphological and biochemical parameters. All apoptotic characters appear stronger and faster in aged cultures than in young, showing increased susceptibility of senescent keratinocytes to PV-IgG-mediated apoptotic death and culture lesions.

Enhancement of Fas-mediated apoptosis in ageing human keratinocytes.

Cellular senescence and apoptosis are two metabolically related and seemingly synergistic processes that are involved in tissue maintenance and homeostasis, anti-tumor protection, and age-related diseases. Despite this apparent co-operativity, senescence can inhibit apoptosis in certain conditions. Here, we describe senescence-apoptosis relationships in human epidermal cells by comparing apoptosis-related effector concentrations in keratinocyte cultures and epidermal skin cells at various stages of ageing.

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins.

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon.

Cellular senescence in human keratinocytes: unchanged proteolytic capacity and increased protein load.

In order to assess the activity of cellular proteasome, we developed a method to permeabilize keratinocyte monolayers and measure proteasome activities intracellularly, using fluorogenic peptide substrates. The observed K(m) did not differ significantly in situ and in soluble extracts, and the K(i) of proteasome inhibitor MG132 was slightly higher in situ (34nM instead of 4nM). Inhibition studies in permeabilized cells showed that MG132 followed competitive inhibition patterns, and clasto-lactacystin beta-lactone non-competitive patterns, as expected.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors.

Conformational and functional properties of an undecapeptide epitope fused with the C-terminal end of the maltose binding protein.

Monoclonal antibody mAb164 is directed against the TrpB2 subunit of the Escherichia coli tryptophan synthase. It recognizes the synthetic peptide P11, constituted of residues 273-283 of TrpB, with high affinity. We constructed a hybrid protein in which the C-terminal end of protein MalE was linked with the N-terminal end of P11.

Recognition of E. coli tryptophan synthase by single-chain Fv fragments: comparison of PCR-cloning variants with the parental antibodies.

The use of a recombinant antibody fragment instead of a complete antibody, as a conformational probe for protein structure and folding studies, can be technically advantageous provided that the recombinant fragment and its parental antibody recognize the antigen through the same mechanism. Monoclonal antibodies mAb19 and mAb93 are directed against the TrpB2 subunit of Escherichia coli tryptophan synthase and they have been extensively used as conformational probes of this protein. DNA sequences coding for single-chain variable fragments (scFv) of mAb19 and mAb93 were cloned and assembled by reverse transcription of the mRNAs from hybridomas and PCR amplification.

Energetic and kinetic contributions of contact residues of antibody D1.3 in the interaction with lysozyme.

Fully functional variable fragments (Fv) of D1.3, a mouse antibody directed against the hen egg lysozyme, were readily produced as hybrids (Fv-MalE) with the maltose-binding protein of Escherichia coli and purified independently of their antigen-binding properties. We used site-directed mutations of residues in the complementarity-determining regions (CDRs) of D1.

Bifunctional hybrids between the variable domains of an immunoglobulin and the maltose-binding protein of Escherichia coli: production, purification and antigen binding.

Hybrids were constructed between the maltose-binding protein of Escherichia coli (MalE) and the variable domains (V-domains) of D1.3, a mouse antibody directed against hen lysozyme. Each V-domain was fused with the C- or N-terminus of MalE and expressed in E.

[Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli].

We have fused the variable domains of a mouse antibody to the C-terminal end of the maltose-binding protein (malE), at the genetic level. The hybrid proteins were expressed in E. coli under control of the malEp promoter, and exported to the periplasm, at low temperature.

Inter-species complementation of a rosy deficiency in Drosophila melanogaster.

A chimeric Xdh gene was constructed in vitro, by recombining DNA sequences from the Dipterans Drosophila melanogaster and Calliphora vicina. The ry506 strain, an eye-colour mutant of Drosophila that is deficient for Xdh, was genetically transformed with the recombinant gene. Transformed flies with ry+ eye phenotype and increased resistance to purine were obtained, showing that the chimeric XDH is physiologically active in Drosophila.

Divergence of the nucleotide sequences encoding xanthine dehydrogenase in Calliphora vicina and Drosophila melanogaster.

We present here two nucleotide sequences, from two different alleles encoding xanthine dehydrogenase in Calliphora vicina. One sequence covers the first exon with 1529 bp upstream from the initial ATG and 1737 bp downstream from the donor end of the first intron. The other sequence starts 2537 bp upstream from the acceptor site of the first intron, and ends 662 bp downstream from the putative polyadenylation site of the transcript.

Conversion and reciprocal exchange between tandem repeats in Drosophila melanogaster.

We have developed an experimental system to assay conversion and reciprocal exchange between tandem repeats in Drosophila melanogaster. In this system, the recombining markers map 0.76 kb apart within the Adh gene, and the length of the repeated unit is 4.

Cloning and partial characterization of the xanthine dehydrogenase gene of Calliphora vicina, a distant relative of Drosophila melanogaster.

In vitro enzymatic assays have shown that an enzyme with typical xanthine dehydrogenase (XDH) activities and electrophoretic mobility slightly different from that of Drosophila XDH is present in Calliphora tissues. A Calliphora genomic sequence has been isolated by low-stringency hybridization to the Drosophila rosy gene (XDH), and partially sequenced. This sequence has been shown to be unique, polymorphic, and it maps on chromosome I.

Identification and molecular analysis of a multigene family encoding calliphorin, the major larval serum protein of Calliphora vicina.

A library of Calliphora vicina genomic DNA was constructed in the lambdaEMBL3 vector and screened for recombinant phages containing chromosomal segments encoding calliphorin, the major larval serum protein (LSP) of Calliphora. A large series of recombinants hybridizing with in vitro labelled poly(A) RNA from Calliphora larval fat bodies and with specific probes derived from the LSP-1 genes of Drosophila melanogaster was isolated. Five of these phages, chosen at random, were shown by hybrid selection to retain calliphorin mRNA specifically.

An efficient program to construct restriction maps from experimental data with realistic error levels.

A novel algorithm has been developed to map restriction fragments, starting from experimental size values with realistic error rates. A high performing PASCAL program has been derived from this algorithm to construct linear maps in minimal computation times.

A directional process of gene conversion is expected to yield dynamic polymorphism associated with stability of alternative alleles in class I histocompatibility antigens gene family.

This work is a contribution to the discussion of the polymorphism of class I histocompatibility antigens (H-2 antigens in the mouse). Specially, the involvement of gene conversion in H-2 polymorphism has been explored through the use of a simplified computer model. It thus appears that in a population of limited size, gene conversion can promote allelic polymorphism while homogenizing the DNA sequences in a multigene family, only if it acts as a directional process.