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. The magnetic cream was applied on the epidermis of human skin organ cultures. The effects on UV-induced cell toxicity, apoptosis and inflammatory cytokine expression were analyzed. A clinical test was performed to check skin moisturization. RESULTS: Nanomagnets were found to be homogenously and stably dispersed. After magnetization, the preparation generated a magnetic field of 1-2 G. Upon cream application, no cytotoxicity and no impairment of cellular vitality were found after 24 and 48 h, respectively. The anti-apoptotic and anti-inflammatory properties of Dermud were not modified, but its long-term effect on moisturization in vivo was slightly increased. CONCLUSION: Nanomagnetic Dermud cream can be used as a tool to analyze the biological effects of nanomagnets dispersed on the skin surface at the cellular and molecular levels, thus allowing to explore the possible therapeutic uses of superficial magnetism for skin care.

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. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.

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. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.

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. Hybrid MalE-P11 was produced in E. coli from a plasmidic gene and purified in one step as MalE. MalE-P11 and the isolated P11 had identical conformational and functional properties according to the following criteria. The NMR spectra of MalE and MalE-P11 in TOCSY experiments showed that the P11 moiety of MalE-P11 moved independently from its MalE moiety. The chemical shifts of the protons for the P11 moiety of MalE-P11 and for the isolated P11 were very close and did not show significant deviations from random coil values. The equilibrium constant of dissociation (KD) from mAb164, measured by a competition ELISA, was identical for MalE-P11 and the isolated P11, around 6 nM. The change of the C-terminal residue of MalE-P11 from Lys into Ala increased 37-fold this dissociation constant. This increase showed that the P11 moiety of MalE-P11 was not degraded. The high molecular mass of MalE-P11 allowed us to follow its kinetics of interaction with immobilized mAb164 by surface plasmon resonance, using the BIAcore apparatus. The rates of association with mAb164 were similar for MalE-P11 and TrpB2, but the dissociation was faster for MalE-P11 than for TrpB2, as previously observed for the isolated P11 by a fluorometric method. Thus, the fusion of peptides with the C-terminal end of MalE could constitute an alternative to chemical synthesis for the study of their recognition by receptors, in vivo or in vitro.

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. A specialized plasmid vector, pFBX, was constructed; it enabled to express the scFvs as hybrids with the maltose-binding protein (MalE) in E. coli, and to purify them by affinity chromatography on cross-linked amylose. Six independent clones were sequenced for each hybridoma. All of them had differences in their nucleotide and amino acid sequences. A competition ELISA and the BIAcore biosensor apparatus were used to compare the energetics and kinetics with which the parental antibodies and the hybrids bound TrpB2. The antigen binding properties of the hybrids were close to those of the parental antibodies and they were only weakly affected by the differences of sequence between the clones, with one exception. The stability of one of the hybrids and its antigen binding properties were strongly modified by a change of Gln6 into Glu, introduced into its VH domain by the PCR primers. Simple models of bimolecular interaction did not fully account for the kinetic profiles obtained with the parental antibodies and the hybrids, and this complexity suggested the existence of a conformational heterogeneity in these molecules.

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 D1.3 as local conformational probes, and compared their effects on the binding of Fv and Fv-MalE to lysozyme. We found that the MalE moiety did not significantly interfere with the interaction between the antigen and the antibody Fv fragment. We then determined the contribution of several potential contact residues of D1.3 in the interaction with lysozyme, by assaying the effect of site-directed mutations on the kinetics of association and dissociation of the complex between Fv-MalE and immobilized lysozyme, using the BIAcore apparatus. While the k(on) values were virtually unaffected by the mutations, the k(off) values varied by more than three orders of magnitude. Both charged (aspartate and arginine) and aromatic (tyrosine and tryptophan) residues in the CDR3 regions of the heavy and light chains of D1.3, which form the center of its antigen-combining site, played a preponderant part in the binding of lysozyme. Our results also showed that indirect hydrogen bonds, bridged by water molecules, contributed significantly to the interaction between D1.3 and lysozyme, and that their energy could be estimated at 1 to 2 kcal.mol-1.

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. coli, either alone or associated with the other V-domain, as a heterodimer (Fv) or as a single-chain fragment (scFv). The hybrids were exported into the bacterial periplasm, purified by affinity chromatography on cross-linked amylose and separated from incomplete products by ion-exchange chromatography. Hybrids between MalE and Fv bound the antigen specifically, with affinities increased up to 10-fold when compared to native D1.3. This strongly suggests that MalE contributed to the binding. The affinities and specificities of the different hybrids, as well as their levels of contamination by incomplete products, depended on their fusion pattern with MalE. Hybrids between MalE and either single V-domain also bound hen lysozyme specifically, which shows that each V-domain can recognize the antigen when fused with MalE. The high affinity of VH-MalE (KD = 3 nM) could be due to both participation of MalE in the binding and a conformational adaptation of the lone V-domain.

[Expression, isolation and purification of antibody fragments fused to maltose-binding protein in Escherichia coli]. / Expression, exportation et purification de fragments d'anticorps fusionnés à la protéine affine du maltose d'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. They were purified by affinity chromatography on cross-linked amylose. When the two variable domains were fused together through a peptide link, the hybrid displayed similar affinity and specificity to the antigen as the native antibody.

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. XDH activity was detected in crude extracts from transformed flies, yet at lower levels than in wild-type controls. The amounts of Xdh transcripts in the transformants were found to be 8 to 16% of the amount of wild-type ry mRNA, suggesting that Calliphora Xdh sequences may be relatively inefficient for mRNA production in Drosophila, or may produce unstable mRNA.

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 4.75 kb. Our results provide a preliminary record of germline frequencies of gene conversion and unequal exchange between these markers. Conversions involving dispersed repeats were not observed, and may be less frequent. This work demonstrates that conversion takes place at an appreciable frequency between tandem repeats in metazoan germline. It confirms that gene conversion can mediate homogenization of reiterated sequences in higher eukaryotes.

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. Comparison with the homologous gene from Drosophila melanogaster (rosy) reveals extensive divergence, with differences in the splicing patterns and no detectable homology between introns or flanking regions. Nevertheless, there is 76% identity between the amino acid (aa) sequences. The pattern of aa differences has been analysed and correlated with predicted three-dimensional (3-D) parameters. These studies consistently suggest that the evolution of the protein was strongly biased for conservation of its 3-D structure. Possible functional significance of the aa changes is discussed.

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. Sequence comparisons provide compelling evidence that it belongs to the XDH gene of Calliphora. Interspecies transformation experiments, aimed at investigating functional as well as structural divergence of the XDH genes of Calliphora and Drosophila, are now possible.

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. Eleven calliphorin mRNA-homologous regions were located on restriction maps of these phages by hybridization with 5' end-labelled poly(A) RNA from Calliphora larval fat bodies. Each phage contains at least two calliphorin genes arranged in direct repeat orientation and seperated by 3.5-5 kb intergenic regions. The genes display similar but not identical restriction patterns. Filter hybridization and heteroduplex analysis indicate that they share a detectable homology with the LSP-1beta gene of D. melanogaster. Whole genome Southern analysis showed that these genes belong to a large family of closely related calliphorin genes which were found by in situ hybridization to polytene chromosomes of trichogen cells to be clustered in region 4a of chromosome 2 of Calliphora vicina.

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. The DNA sequences of the preferential targets are then polymorphic and evolutionary versatile, while other genes of the family mostly behave as sequence donors, have more stable DNA sequences and are much less polymorphic. The characteristics of such a polymorphism generator are discussed with respect to their functional and evolutionary implications.

Comparison of nucleotide sequences of mRNAs belonging to the mouse H-2 multigene family.

The complete nucleotide sequences of three cDNAs coding for the C-terminal part of mouse histocompatibility (H-2) antigens, and for the 3' non coding regions of these clones have been determined. Comparison of the sequence indicates a large homology throughout the coding and non-coding regions and suggests the existence of a genetic mechanism which homogenizes nucleotide sequences among genes of the H-2 multigene family.

Molecular cloning of the genome of poliovirus type 1.

Poliovirus cDNA.RNA hybrids were prepared from the Mahoney strain of poliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coli plasmid pBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase T1-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kilobase pairs, originated from various parts of the viral RNA, covering at least 99% of the genome length. Due to overlapping of the clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPV1-366.

DNA organisation in the chicken lysozyme gene region.

DNA sequences surrounding the lysozyme gene of the chicken have been cloned in several recombinants which define a region of 40 Kb. We have detected no other gene with a sequence related to that of the lysozyme gene, nor any gene expressed in the oviduct in these recombinants. This situation contrasts with that of the ovalbumin gene, in the vicinity of which lie two other genes of related structure expressed in the oviduct under hormonal control. The lysozyme gene region, however contains a complex array of repeated sequences, which have been resolved into at least five classes. An inverted repeat overlaps the lysozyme gene itself.