Melanosome Capping of Keratinocytes in Pigmented Reconstructed Epidermis – Effect of Ultraviolet Radiation and 3‐Isobutyl‐1‐Methyl‐Xanthine on Melanogenesis

Reconstructed pigmented epidermis was established by co‐seeding autologous melanocytes and keratinocytes onto a dermal substrate and culturing for up to 6 weeks at the air–liquid interface. Inspection of the tissue architecture revealed that melanocytes are regularly interspersed only in the basal layer and transfer melanosomes to the keratinocytes. We report for the first time, the in vitro formation of supranuclear melanin caps above the keratinocyte nuclei. The formation and abundance of these melanin caps could be enhanced by pigment modifiers such as ultraviolet light and 3‐isobutyl‐1‐methyl‐xanthine (IBMX). In untreated cultures, the capping was observed in the spinous layers after 6 weeks of culture, whereas after irradiation or supplementation of the culture medium with IBMX, the capping occurred already in the basal layer 2 weeks after initiation of the stimulus. In this study, we show that IBMX and ultraviolet irradiation stimulate pigmentation via different mechanisms. After supplementation of the culture medium with IBMX the increase in pigmentation was entirely due to the increase in melanocyte activity as observed by increased dendrite formation, melanin production and transport to the keratinocytes and was not due to an increase in melanocyte proliferation. In contrast, after UV irradiation, the increase in pigmentation was also accompanied with an increase in melanocyte proliferation as well as an increase in melanocyte activity. In conclusion, we describe the establishment of pigmented reconstructed epidermis with autologous keratinocytes and melanocytes that can be kept in culture for a period of at least 6 weeks. The complete program of melanogenesis occurs: melanosome synthesis, melanosome transport to keratinocytes, supranuclear capping of keratinocyte nuclei and tanning of the epidermis. This enables sustained application of pigment stimulators over a prolonged period of time and also repeated application of pigment stimulators to be studied.     

Assembly,Target‐Signaling and Intracellular Transport of Tyrosinase Gene Family Proteins in the Initial Stage of Melanosome Biogenesis

Assembly, target‐signaling and transport of tyrosinase gene family proteins at the initial stage of melanosome biogenesis are reviewed based on our own discoveries. Melanosome biogenesis involves four stages of maturation with distinct morphological and biochemical characteristics that reflect distinct processes of the biosynthesis of structural and enzymatic proteins, subsequent structural organization and melanin deposition occurring in these particular cellular compartments. The melanosomes share many common biological properties with the lysosomes. The stage I melanosomes appear to be linked to the late endosomes. Most of melanosomal proteins are glycoproteins that should be folded or assembled correctly in the ER through interaction with calnexin, a chaperone associated with melanogenesis. These melanosomal glycoproteins are then accumulated in the trans Golgi network (TGN) and transported to the melanosomal compartment. During the formation of transport vesicles, coat proteins assemble on the cytoplasmic face of TGN to select their cargos by interacting directly or indirectly with melanosomal glycoproteins to be transported. Adapter protein‐3 (AP‐3) is important for intracellular transport of tyrosinase gene family proteins from TGN to melanosomes. Tyrosinase gene family proteins possess a di‐leucine motif in their cytoplasmic tail, to which AP‐3 appears to bind. Thus, the initial cascade of melanosome biogenesis is regulated by several factors including: 1) glycosylation of tyrosinase gene family proteins and their correct folding and assembly within ER and Golgi, and 2) supply of specific signals necessary for intracellular transport of these glycoproteins by vesicles from Golgi to melanosomes.     

Plasticity of Cadherin–Catenin Expression in the Melanocyte Lineage

Cadherins are calcium‐dependent cell adhesion receptors with strong morphoregulatory functions. To mediate functional adhesion, cadherins must interact with actin cytoskeleton. Catenins are cytoplasmic proteins that mediate the interactions between cadherins and the cytoskeleton. In addition to their role in cell–cell adhesion, catenins also participate in signaling pathways that regulate cell growth and differentiation. Cadherins and catenins appear to be involved in melanocyte development and transformation. Here, we investigated the function of cadherin–catenin complexes in the normal development and transformation of melanocytes by studying the patterns of expression of the cell–cell adhesion molecules, E‐, N‐ and P‐cadherin, and the expression of their cytoplasmic partners, α‐, β‐ and Γ‐catenin, during murine development. Similar analyses were performed in vitro using murine melanoblast, melanocyte, and melanoma cell lines in the presence and absence of keratinocytes, the cells with which melanocytes interact in vivo. Overall, the results suggest that the expression of cadherins and catenins is very plastic and depends on their environment as well as the transformation status of the cells. This plasticity is important in fundamental cellular mechanisms associated with normal and pathological ontogenesis, as well as with tumorigenesis.     

Co‐Culture of Mouse Epidermal Cells for Studies of Pigmentation

Interactions between melanocytes and keratinocytes in the skin suggest bi‐directional interchanges between these two cell types. Thus, melanocytes cultured alone may not accurately reflect the physiology of the skin and the effects of physiological regulators in vivo, because they do not consider possible interactions with keratinocytes. As more and more pigment genes are identified and cloned, the characterization of their functions becomes more of a challenge, particularly with respect to their roles in the processing and transport of melanosomes and their transfer to keratinocytes. Immortalized melanocytes mutant at these loci are now being routinely generated from mice, but interestingly, successful co‐culture of murine melanocytes and keratinocytes is very difficult compared with their human counterparts. Thus, we have now optimized co‐culture conditions for murine melanocytes and keratinocytes so that pigmentation and the effects of specific mutations can be studied in a more physiologically relevant context.     

The Expression and Activation of Protease‐Activated Receptor‐2 Correlate with Skin Color

Skin color results from the production and distribution of melanin in the epidermis. The protease‐activated receptor‐2 (PAR‐2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR‐2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color‐dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR‐2 may play a role in the modulation of pigmentation in a skin type‐dependent manner. We examined the expression of PAR‐2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR‐2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR‐2‐specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR‐2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin‐derived keratinocytes. These results demonstrate that PAR‐2 expression and activity correlate with skin color, suggesting the involvement of PAR‐2 in ethnic skin color phenotypes.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Exchange of Microtubule Molecular Motors During Melanosome Transport in <Emphasis Type="Italic">Xenopus laevis</Emphasis> Melanophores is Triggered by Collisions with Intracellular Obstacles

The observation that several cargoes move bidirectionally along microtubules in vivo raised the question regarding how molecular motors with opposed polarity coordinate during transport. In this work, we analyzed the switch of microtubule motors during the transport of melanosomes in Xenopus melanophores by registering trajectories of these organelles moving along microtubules using a fast and precise tracking method. We analyzed in detail the intervals of trajectories showing reversions in the original direction of transport and processive motion in the opposite direction for at least 250 nm. In most of the cases, the speed of the melanosome before the reversion slowly decreases with time approaching zero then, the organelle returns over the same path moving initially at a very high speed and slowing down with time. These results could be explained according to a model in which reversions are triggered by an elastic collision of the cargo with obstacles in the cytosol. This interaction generates a force opposed to the movement of the motor-driven organelle increasing the probability of detaching the active motors from the track. The model can explain reversions in melanosome trajectories as well as other characteristics of in vivo transport along microtubules observed by other authors. Our results suggest that the crowded cytoplasm plays a key role in regulating the coordination of microtubules-dependent motors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

During Human Melanoma Progression AP‐1 Binding Pairs are Altered with Loss of c‐Jun In Vitro

We demonstrated previously that c‐Jun, JunB and c‐Fos RNA were dysregulated in metastatic melanoma cells compared with normal human melanocytes. The purpose of this study was to evaluate the distribution in composition of AP‐1 dimers in human melanoma pathogenesis. We investigated AP‐1 dimer pairing in radial growth phase‐like (RGP) (w3211) and vertical growth phase‐like (VGP) (w1205) human melanoma cells and metastatic cell lines (cloned from patients, c83‐2c, c81‐46A, A375, respectively) compared with melanocytes using electrophoretic mobility shift assay (EMSA), Western blot and transfection analyses. There are progressive variations in AP‐1 composition in different melanoma cell lines compared with normal melanocytes, in which c‐Jun, JunD and FosB were involved in AP‐1 complexes. In w3211, c‐Jun, JunD and Fra‐1 were involved in AP‐1 binding, while in w1205, overall AP‐1 binding activity was decreased significantly and supershift binding was detected only with JunD antibodies. In metastatic c81‐46A and A375 cells, only JunD was involved in AP‐1 binding activity, but in a third (c83‐2c) c‐Jun, JunD and Fra‐1 were present. Western blot evaluation detected c‐Jun in melanocytes and w3211, but this component was decreased significantly or was not detectable in w1205, c81‐46A and A375 cells. In contrast, JunD protein was elevated in c81‐46A and c83‐2c cells compared with melanocytes and RGP and VGP cell lines. Normal melanocytes and c83‐2c cells (which have c‐Jun involved in AP‐1 binding), transfected with c‐Jun antisense and treated with cisplatin, showed higher viability compared with untransfected cells, while in c81‐46A cells (in which only JunD is detectable) no change in cell viability was observed following treatment with cisplatin and c‐jun antisense transfection. A dominant‐negative c‐Jun mutant (TAM67) significantly increased the soft agar colony formation of w3211 and c83‐2c cells. These results suggest that components of AP‐1, especially c‐Jun, may offer a new target for the prevention or treatment of human melanoma progression.     

Synthesis of Novel Diselenide‐Linked Porphyrin Dimers under Phase‐Transfer Catalysis Condition and Their Interactions with DNA

Novel diselenide‐linked porphyrin dimers were synthesized under phase‐transfer catalysis conditions. The targeted compounds were characterized by ¹H‐NMR, high‐resolution mass spectrometry, UV/VIS and fluorescence spectroscopies, redox‐potential measurements, and elemental analysis. The interaction of the title compounds with DNA was studied using UV/VIS, fluorescence, and circular dichroism (CD) spectroscopies. The relative rates of singlet‐oxygen production from the diselenide‐linked porphyrin dimers upon photoirradiation were also measured.     

Phosphorylation of Dab2 is involved in inhibited VEGF‐VEGFR‐2 signaling induced by downregulation of syndecan‐1 in glomerular endothelial cell

Disabled‐2 (Dab2) and PAR‐3 (partitioning defective 3) are reported to play critical roles in maintaining retinal microvascular endothelial cells biology by regulating VEGF‐VEGFR‐2 signaling. The role of Dab2 and PAR‐3 in glomerular endothelial cell (GEnC) is unclear. In this study, we found that, no matter whether with vascular endothelial growth factor (VEGF) treatment or not, decreased expression of Dab2 could lead to cell apoptosis by preventing activation of VEGF‐VEGFR‐2 signaling in GEnC, accompanied by reduced membrane VEGFR‐2 expression. And silencing of PAR‐3 gene expression caused increased apoptosis of GEnC by inhibiting activation of VEGF‐VEGFR‐2 signaling and membrane VEGFR‐2 expression. In our previous research, we found that the silencing of syndecan‐1 gene expression inhibited VEGF‐VEGFR‐2 signaling by modulating internalization of VEGFR‐2. And our further research demonstrated that downregulation of syndecan‐1 lead to no significant change in the expression of Dab2 and PAR‐3 both at messenger RNA and protein levels in GEnC, while phosphorylation of Dab2 was significantly increased in GEnC transfected with Dab2 small interfering RNA (siRNA) compared with control siRNA. Atypical protein kinase C (aPKC) could induce phosphorylation of Dab2, thus negatively regulating VEGF‐VEGFR‐2 signaling. And we found that decreased expression of syndecan‐1 lead to activation of aPKC, and aPKC inhibitor treatment could block phosphorylation of Dab2 in GEnC. Besides, aPKC inhibitor treatment could activate VEGF‐VGEFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA in a dose‐dependent manner. In conclusion, we speculated that phosphorylation of Dab2 is involved in preventing activation of VEGF‐VEGFR‐2 signaling in GEnC transfected with syndecan‐1 siRNA. This provides a new target for the therapy of GEnC injury and kidney disease.     

Charge‐Transfer States at Organic–Organic Interfaces: Impact of Static and Dynamic Disorders

Molecular dynamics simulations are combined with density functional theory calculations to evaluate the impact of static and dynamic disorders on the energy distribution of charge‐transfer (CT) states at donor–acceptor heterojunctions, such as those found in the active layers of organic solar cells. It is shown that each of these two disorder components can be partitioned into contributions related to the energetic disorder of the transport states and to the disorder associated with the hole–electron electrostatic interaction energies. The methodology is applied to evaluate the energy distributions of the CT states in representative bulk heterojunctions based on poly‐3‐hexyl‐thiophene and phenyl‐C₆₁‐butyric‐acid methyl ester. The results indicate that the torsional fluctuations of the polymer backbones are the main source of both static and dynamic disorders for the CT states as well as for the transport levels. The impact of static and dynamic disorders on radiative and nonradiative geminate recombination processes is also discussed.     

The Roles of Structural Order and Intermolecular Interactions in Determining Ionization Energies and Charge‐Transfer State Energies in Organic Semiconductors

The energy landscape in organic semiconducting materials greatly influences charge and exciton behavior, which are both critical to the operation of organic electronic devices. These energy landscapes can change dramatically depending on the phases of material present, including pure phases of one molecule or polymer and mixed phases exhibiting different degrees of order and composition. In this work, ultraviolet photoelectron spectroscopy measurements of ionization energies (IEs) and external quantum efficiency measurements of charge‐transfer (CT) state energies (E_CT) are applied to molecular photovoltaic material systems to characterize energy landscapes. The results show that IEs and E_CT values are highly dependent on structural order and phase composition. In the sexithiophene:C₆₀ system both the IEs of sexithiophene and C₆₀ shift by over 0.4 eV while E_CT shifts by 0.5 eV depending on molecular composition. By contrast, in the rubrene:C₆₀ system the IE of rubrene and C₆₀ vary by ≤ 0.11 eV and E_CT varies by ≤ 0.04 eV as the material composition varies. These results suggest that energy landscapes can exist whereby the binding energies of the CT states are overcome by energy offsets between charges in CT states in mixed regions and free charges in pure phases.     

Regulation of protease‐activated receptor‐2 expression in gingival fibroblasts and Jurkat T cells by Porphyromonas gingivalis

Periodontal disease destroys the tooth‐supporting tissues as a result of chronic inflammation elicited by bacterial accumulation on tooth surfaces. Porphyromonas gingivalis is a major periodontal pathogen, with a significant capacity to perturb connective tissue homeostasis and immune responses in the periodontium, attributed to its virulence factors, including a group of secreted cysteine proteases (gingipains). PAR‐2 (protease‐activated receptor‐2) is a G‐protein‐coupled receptor activated upon proteolytic cleavage, mediating intracellular signalling events related to infection and inflammation, such as cytokine production. GF (gingival fibroblasts) and T cells have central roles in periodontal inflammation, which can potentially be mediated by PAR‐2. The aims of this study were to investigate the effects of P. gingivalis on PAR‐2 gene expression in human GF and Jurkat T cells, using quantitative real‐time PCR, and to evaluate the involvement of gingipains. After 6 h of challenge with ascending concentrations of P. gingivalis, PAR‐2 expression was up‐regulated in both cell types by approximately 5‐fold, compared with the control. The P. gingivalis concentration required for maximal PAR‐2 induction was 4‐fold greater in GF than Jurkat T cells. Heat inactivation or chemical inhibition of cysteine proteases abolished the capacity of P. gingivalis to induce PAR‐2 expression in Jurkat T cells. In conclusion, P. gingivalis can induce PAR‐2 expression in GF and Jurkat T cells, potentially attributed to its gingipains. These findings denote that P. gingivalis may perturb the host immune and inflammatory responses by enhancing PAR‐2 expression, thus contributing to the pathogenesis of periodontal disease.     

Charge Transfer Modulated Activity of Carbon‐Based Electrocatalysts

Electrocatalysis is the most important electrode reactions for many energy storage and conversion devices, which are considered a key part of the resolution of the energy crisis. Toward this end, design of efficient electrocatalysts is of critical significance. While extensive research has been extended to develop excellent electrocatalysts, the fundamental understanding of the relationship between the electronic and structural properties of electrocatalysts and the catalytic activity must remain a priority. In this review, the activity modulation of electrocatalysts by charge transfer effects, including intramolecular and intermolecular charge transfer, is systematically introduced. With suitable charge transfer modification, such as heteroatom doping, defect engineering, molecule functionalization, and heterojunctions, the electrocatalytic activity of carbon‐based electrocatalysts can be significantly boosted. The manipulation of the electronic structure of carbon‐based materials by charge transfer may serve as a fundamental mechanism for performance enhancement. After establishing an understanding of the relationship between catalytic activity and charge transfer, the opportunities and challenges for the design of electrocatalyst with charge transfer effects are discussed.