MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms.

Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure.

Constitutive expression of MC1R in HaCaT keratinocytes inhibits basal and UVB-induced TNF-alpha production.

Alpha-melanocyte stimulating hormone (alpha-MSH) binds to melanocortin-1 receptor (MC1R) on melanocytes to stimulate pigmentation and modulate various cutaneous inflammatory responses. MC1R expression is not restricted to melanocytic cells and may be induced in keratinocytes after UVB exposure. We hypothesized that MC1R signaling in keratinocytes, wherein basal conditions are barely expressed, may modulate mediators of inflammation, such as nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor-alpha (TNF-alpha).

In vitro stimulation of synthesis of key DEJ constituents in a reconstructed skin model: a quantitative study.

Fighting skin ageing is one of the major targets of cosmetology research. However, traditional approaches to skin ageing using stimulation of basal keratinocyte proliferation and fibroblastic neosynthesis appear today to be incomplete, particularly considering changes occurring at the dermal-epidermal junction (DEJ) during the course of ageing. Unfortunately, the lack of in vitro model limits the exploration process of the phenomena of DEJ ageing, and particularly the evaluation of the changes of key components, that are laminin-5, types IV and VII collagens.

An optimized method for intensive screening of molecules that stimulate beta-defensin 2 or 3 (hBD2 or hBD3) expression in cultured normal human keratinocytes.

Normal human skin controls the intrusion of microorganisms by the production of peptide antibiotics such as defensins. The aim of our study was to develop a culture model of normal human keratinocytes for optimal beta-defensin mRNA detection which allows the screening of molecules able to stimulate hBD2 and hBD3 without inducing pro-inflammatory cytokines. A keratinocyte culture model in 96-well plates, in high calcium medium (1.

Differential effects of allergens and irritants on early differentiating monocyte-derived dendritic cells.

Phenotypic modifications induced by contact allergens on monocyte-derived dendritic cells (MoDC) have been proposed as an in vitro alternative method to discriminate potential sensitizers from irritants. However, the sensitivity of the assay remains controversial. In all the studies reported so far, DC treatment with chemicals was carried out after 5 to 6 days of monocyte culture.

Cell surface expression of melanocortin-1 receptor on HaCaT keratinocytes and alpha-melanocortin stimulation do not affect the formation and repair of UVB-induced DNA photoproducts.

Ultraviolet (UV) exposure induces an up-regulation of melanocortin-1 receptor (MC1R) expression in human skin and the alpha-melanocyte-stimulating hormone (alpha-MSH) may reduce UVB-induced DNA damage in normal human melanocytes. Using high-performance liquid chromatography coupled to tandem mass spectrometry, we investigated the formation and repair of DNA lesions in UVB-irradiated HaCaT cells stably transfected with the wild type MC1R gene (HaCaT-MC1R). Similar levels of 8 bipyrimidine photoproducts including cyclobutane pyrimidine dimers (CPDs) (T<>T, T<>C, C<>T), (6-4) photoproducts ((6-4)PPs) (TT-(6-4)PPs, TC-(6-4)PPs) and their Dewar valence isomers together with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were found to be generated in both non-transfected and HaCaT-MC1R cells after UVB exposure.

Moderate skin sensitizers can induce phenotypic changes on in vitro generated dendritic cells.

In the present study, we analyzed the phenotypic alterations induced by several allergens on immature dendritic cells (DC), with the aim to develop a potential in vitro alternative for predicting the sensitizing potential of chemicals. DC were generated from human monocytes cultured in the presence of GM-CSF, IL-4 and TGF-beta1 and treated for 2 or 4 days with different chemicals. Surface marker expression (HLA-DR, CD1a, CD40, CD54, CD83, CD86, CCR7 and E-cadherin) was analyzed by flow cytometry.

Adipocyte differentiation of multipotent cells established from human adipose tissue.

In this study multipotent adipose-derived stem cells isolated from human adipose tissue (hMADS cells) were shown to differentiate into adipose cells in serum-free, chemically defined medium. During the differentiation process, hMADS cells exhibited a gene expression pattern similar to that described for rodent clonal preadipocytes and human primary preadipocytes. Differentiated cells displayed the key features of human adipocytes, i.

Calcium triggers beta-defensin (hBD-2 and hBD-3) and chemokine macrophage inflammatory protein-3 alpha (MIP-3alpha/CCL20) expression in monolayers of activated human keratinocytes.

The inducible epidermal beta-defensins and the chemokine macrophage inflammatory protein-3alpha (MIP-3alpha/CCL20) are important mediators involved in innate and adaptive immunity and in the recruitment of immune cells. The aim of our study was to determine whether calcium could trigger the induction of beta-defensins (hBD-2 and hBD-3) mRNA and the release of MIP-3alpha by normal human keratinocyte monolayers. Epidermal cells derived from foreskin were cultured in defined medium supplemented with different calcium levels (0.

Measurement of epidermal moisture content by magnetic resonance imaging: assessment of a hydration cream.

The moisture content of the epidermis was measured by magnetic resonance imaging (MRI), using transverse relaxation time. The spatial resolution was 86 microns, allowing a quantitative, accurate and localized determination of variations in epidermal hydration. The wrists of 15 volunteers were studied before and after application of a hydration cream.

2D NMR studies and 3D structure of the parallel-stranded duplex oligonucleotide Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements.

Solution structure of the parallel-stranded duplex oligonucleotide alpha-d(TCTAAAC)-beta-d(AGATTTG) via complete relaxation matrix analysis of the NOE effects and molecular mechanics calculations.

The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion.

NMR studies of complex formation between the modified oligonucleotide d(T*TCTGT) covalently linked to an acridine derivative and its complementary sequence d(GCACAGAA).

The oligodeoxynucleotide d(TTCTGT) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phOsphate via a pentamethylene linker (m5). In order to avoid its hydrolysis by nucleases inside the cel., one of its phosphates (TpT) was substituTed with a neopentyl group.

2D-NMR studies of the unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG).

The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG).

Interaction between the different domains of aminoacyl-tRNA and the elongation-factor-Tu x kirromycin complex.

In this work, we have studied the effect of aa-tRNA and derived 3' aminoacylated fragments on the EF-Tu GTPase in the presence of kirromycin, using two systems: without and with ribosomes. The aa-tRNA fragments were obtained by enzymatic digestion. Procedures for the enzymatic preparation of C-A-Val and Val-tRNA Val1 3' half molecule, as well as a purification method for short 3' aminoacylated fragments based on the amino group charge, were newly developed for this work.

Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu.

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule.