Project description:Epidermal melanocytes form synaptic-like contacts with cutaneous nerve fibers but the functional outcome of this connection remains elusive. In this pilot study, we used our fully humanized re-innervated skin organ culture model to investigate whether melanocyte-nerve fibers interaction play a role in UV-B-induced melanogenesis. UV-B-irradiation significantly enhanced melanin content and tyrosinase activity in re-innervated skin ex vivo compared to non-innervated controls, indicating that neuronal presence is essential for exacerbating pigmentation upon UV-B irradiation in long-term culture. Comparative transcriptomic analysis between laser-capture-microdissected melanocytes from freshly embedded human skin and published microarray data on in vitro primary melanocytes identified Semaphorin-4A (SEMA4A) as possible mediator of melanocyte-nerve fibers interactions. SEMA4A protein levels in Gp100+ epidermal melanocytes were significantly higher in re-innervated skin than in skin alone, and reduced by UV-B treatment. Analysis of melanocytes in vitro showed reduced SEMA4A protein expression 24h after UV-B-irradiation while SEMA4A secretion into the medium was increased. In sensory neurons, conditioned media (CM) from UV-B irradiated melanocytes stimulated tubulin expression and axon growth. Re-transfer of CM from neurons that received CM from melanocytes back to melanocytes resulted into significant increased melanin content only when the CM derived from neurons previously receiving CM from UV-B irradiated melanocytes. These findings highlight the importance of melanocyte-neuron interactions for UV-B-induced melanogenesis and suggest that secreted proteins (e.g. SEMA4A) can function as a novel target in the treatment of hypo- and hyperpigmentation disorders.
Project description:UV-induced CPDs were mapped in primary skin melanocytes or normal human skin fibroblasts following either UVC or UVB irradiation and in isolated human genomic DNA (naked DNA control) that was UVB or UVC irradiated. CPDs were mapped across the human genome using the CPD-capture-seq method and the resulting libraries were captured for ~4000 genomic regions of interest (~3 Mbp) of the human genome by the company Rapid Genomics prior to Illumina sequencing
Project description:We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after UVB at different time points (6, 12 and 24h) to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of ultraviolet-B radiation (UVB). Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. We analysed six lines of melanocytes isolated from lightly pigmented neonatal foreskin (LM), and six lines from darkly pigmented neonatal foreskin (DM), at 0, 6, 12 and 24 hours post UV irradiation. We also assessed the effect of different keratinocyte-conditioned media (KCM+ or KCM-)
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Gene methylation profiling of immortalized human mesenchymal stem cells comparing HPV E6/E7-transfected MSCs cells with human telomerase reverse transcriptase (hTERT)- and HPV E6/E7-transfected MSCs. hTERT may increase gene methylation in MSCs. Goal was to determine the effects of different transfected genes on global gene methylation in MSCs.