Human dermal papilla (DP) cells grown in twodimensional (2D) culture have been studied extensively. However, key differences exist between DP cell activities in vivo and in vitro. Using a suspension method of cell culture to maintain DP cells, we created three-dimensional (3D) dermal spheres morphologically akin to intact (anagen) DPs. Analysis of these spheres using immunocytochemistry demonstrates that they have expression profiles different from papilla cells cultured in 2D but with many similarities to intact DPs. This method of DP cell culture may provide us with a tool to elucidate our understanding of signalling within the DP as it relates to induction, maintenance or even inhibition of hair growth.

A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.

As a first line of defence, the skin is equipped with a complex and interactive nerve fibre system to detect irritants and maintain homeostasis. The dermal component of this fibre network has been well characterized and fibres are known to extend throughout the viable epidermis as free nerve endings. To date, this epidermal component remains poorly characterized. We have visualized human volar forearm epidermal nerve fibres by laser-scanning confocal microscopy using the pan-neuronal marker, protein gene-product 9.5 and specific antibodies to substance P. calcitonin gene-related peptide and nerve growth factor. In addition to the varicose free nerve endings, there is a 3-D fibre network in normal human epidermis, with frequent branching of fibres. Branching can be seen to converge on a central trunk apparently extending to the dermis. Thin unmyelinated fibres can be seen in all layers of the viable epidermis. Substance P staining is rarely observed and is much less intense than the protein gene-product 9.5 staining. Calcitonin gene-related peptide and nerve growth factor were not detected in volar forearm epidermis by this method. Pretreatment of the skin in vivo with the neuropharmacological agent, capsaicin, resulted in loss of epidermal fibre staining indicating that these are sensory fibres of the primary C-afferent type. Epidermal innervation in racial and ethnic skin types was also assessed. No apparent difference in innervation was observed between European caucasian and Japanese/Chinese skin at the architectural or biochemical level, i.e. the presence, properties and biochemical content of fibres was similar in all cases tested.

Crystals of partially neutralized stearic acid with triethanolamine (TEA) were prepared by mixing these two materials above 80 degrees C and then cooling. The crystalline composition and the structure and melting behavior of the resultant products were characterized with small-angle and wide-angle X-ray diffraction, thermal analysis, microscopy, and infrared spectroscopy. It was discovered that an acid-soap complex of 2:1 fixed stoichiometric ratio exists between stearic acid and TEA stearate. A binary phase diagram of stearic acid and TEA soap is built based on the experimental results; this is the first published record of a binary phase diagram for amine-based soap. Its behavior is significantly different from that of binary systems of fatty acid and alkali soap.

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