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Recently, alpha6 integrin has been proposed as the epithelial cell receptor for papillomavirus. This study investigated whether alpha6 integrin is the cellular receptor for bovine papillomavirus type 4 (BPV-4), which is strictly epitheliotropic and infects the mucous epithelium of the upper digestive tract. Primary bovine mucosal keratinocytes from the palate of a foetus (PalK) displayed high levels of alpha6 integrin; matched primary fibroblasts from the same biopsy (PalF) expressed almost no alpha6 integrin. However, BPV-4 bound both PalK and PalF to similar, saturable levels. Native BPV-4 virions infected PalK in vitro, as detected by RT-PCR of E7 RNA. Infection could be blocked by excess virus-like particles (VLPs) and by neutralizing antisera against L1-L2 and L1 VLPs or by denaturation of the virions, supporting the view that infection in vitro mimics the process in vivo. alpha6 integrin-negative human keratinocyte cell lines were derived from patients affected by junctional epidermolysis bullosa presenting genetic lesions in their hemidesmosomes. The level of alpha6 integrin expression was determined in these cell lines by in situ immunofluorescence and FACS. Despite the absence of alpha6 integrin expression by BO-SV cells, they were bound by BPV-4 to similar, saturable levels as normal keratinocytes, KH-SV. Furthermore, BO-SV and KH-SV cells were both infected by BPV-4 to apparently the same extent as PalK cells. These results are consistent with the conclusion that alpha6 integrin is not the obligatory receptor for a bovine mucosotropic papillomavirus.

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Ultraviolet (UV) radiation is the main physiological stimulus for human skin pigmentation. Within the epidermal-melanin unit, melanocytes synthesize and transfer melanin to the surrounding keratinocytes. Keratinocytes produce paracrine factors that affect melanocyte proliferation, dendricity, and melanin synthesis. In this report, we show that normal human keratinocytes secrete nitric oxide (NO) in response to UVA and UVB radiation, and we demonstrate that the constitutive isoform of keratinocyte NO synthase is involved in this process. Next, we investigate the melanogenic effect of NO produced by keratinocytes in response to UV radiation using melanocyte and keratinocyte cocultures. Conditioned media from UV-exposed keratinocytes stimulate tyrosinase activity of melanocytes. This effect is reversed by NO scavengers, suggesting an important role for NO in UV-induced melanogenesis. Moreover, melanocytes respond to NO-donors by decreased growth, enhanced dendricity, and melanogenesis. The rise in melanogenesis induced by NO-generating compounds is associated with an increased amount of both tyrosinase and tyrosinase-related protein 1. These observations suggest that NO plays an important role in the paracrine mediation of UV-induced melanogenesis.

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Ultraviolet B (UVB) radiation is the main physiological stimulus for human skin pigmentation; however, the molecular mechanisms underlying this process are still unclear. Recently, nitric oxide (NO) and cGMP have been involved in mediation of skin erythema induced by UVB. Therefore, we investigated the role of NO and cGMP in UVB-induced melanogenesis. In this study, we demonstrated that UVB stimulation of melanogenesis was mimicked by exogenous NO donors. Additionally, we showed that NO stimulated cGMP synthesis and that cGMP was also a potent stimulator of melanogenesis. Furthermore, the inhibition of the melanogenic effect of NO by guanylate cyclase inhibitor demonstrated that NO mediated its effect through the activation of guanylyl cyclase. Interestingly, 1 min after UVB irradiation, we observed a significant increase in cGMP content in melanocytes. The effects of UVB on cGMP production and on melanogenesis were blocked by both guanylate cyclase and NO synthase inhibitors. Additionally, inhibition of cGMP-dependent kinase also prevented the stimulation of melanogenesis by UVB and NO. Therefore, we concluded that NO and cGMP production is required for UVB-induced melanogenesis and that cGMP mediated its melanogenic effects mainly through the activation of cGMP-dependent kinase.

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The effect of a light stable retinoid (CD 367) was studied on sea urchin embryos. CD 367 did not affect sperm-egg interaction. In a range of concentrations between 10 and 100 microM, CD 367 delayed the first and the second cleavages. When added after fertilization, micromolar amounts of CD 367 delayed hatching and produced embryonic abnormalities in a dose-dependent manner. Mesodermal cells, primary (PMC) and secondary (SMC) mesenchyme cells migration was particularly disturbed, leading to exogastrulations and calcified spicules malformations. Concentrations of CD 367 higher than 8 microM were embryolethal. Micromolar amount of CD 367 increased plasmalemma Ca2+ permeability of fertilized eggs but not of unfertilized eggs. CD 367 inhibited ATP-dependent intracellular sequestration of Ca2+ in a range of concentrations similar to those affecting egg cleavage and embryonic structures. Since we were unable to detect nuclear receptors for CD 367 in sea urchin eggs and ovocytes, these effects probably are not related to interaction of the retinoid with members of the RAR family, to which CD 367 has a high affinity, but rather to its toxicity by the means of some unknown mechanisms.

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The effect of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a polychlorinated herbicide, on the early development of sea urchin eggs and on Ca(2+) permeability was investigated. Concentrations lower than 5 x 10(-4)m delayed the first cleavages and produced a teratogenic effect characterized by a large spectrum of structural malformations at the pluteus stage. The cleaving stage (pre-hatching) was the period most sensitive to the compound. Upper concentrations caused a stepwise dose-dependent lethality associated with arrest of cleavage. 2,4,5-T increased plasmalemma Ca(2+) permeability of unfertilized eggs by opening voltage-dependent Ca(2+) channels; verapamil (10(-4)m) and nifedipine (10(-4)m) abolished this effect. Ca(2+) permeability was also increased by 2,4,5-T after fertilization of the eggs. ATP-dependent intracellular sequestration of Ca(2+), measured in isolated cortices, was inhibited by 2,4,5-T. Ca(2+) movement was affected over a range of concentrations similar to those producing embryonic abnormalities and lethality. The results suggest that the teratogenic potency of 2,4,5-T is associated with delay in first cleavages and alterations in Ca(2+) homoeostasis.

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The effect of direct (chlorambucil and allopurinol) and indirect (cyclophosphamide) teratogens on the fertilization and early development of sea urchin embryos has been investigated. Fertilization was affected by none of the drugs tested. Continuous exposure of embryos to chlorambucil (10(-6) to 3 x 10(-4) M) starting after fertilization delayed the first cleavage and hatching. Developmental defects in chlorambucil-treated embryos consisted mainly of blastula and gastrula-arrested embryos and in a limited number (25%) of plutei with malformed gut or skeleton. Post-hatching exposure to chlorambucil led to malformed plutei only. Early (pre-hatching) exposure to allopurinol (10(-6) to 10(-3) M) did not affect cleavage but induced developmental defects in a ratio comparable to chlorambucil. Post-hatching exposure to allopurinol failed to affect the embryogenesis. The indirect teratogen cyclophosphamide (10(-6) to 3 x 10(-5) M) had no effect on the early embryogenesis. Results were discussed in view of using sea urchin embryos to detect and analyze the early mechanisms of teratogenic action.

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Crassolide, a monocyclic diterpene isolated and purified from the soft coral Lobophytum crassum, inhibited the cell cleavage of sea urchin eggs without affecting fertilization. The effect was observed with concentrations above 2 x 10(-5)m in egg suspensions. Addition of crassolide between 5 and 40 min post-fertilization totally blocked the first cleavage, which in the control occurs 1 hr after fertilization. When added between 50 and 60 min post-fertilization, crassolide produced polynucleated cells in embryos. Crassolide did not affect the egg permeability to Na(+) and Ca(2+), but caused an increase of 0.2 units in the intracellular pH of fertilized eggs coupled with a proton efflux. Crassolide, which does not affect Ca(2+) influx or permeability at the level of storage in reticular vesicles, could be used as a negative control when analysing calcium changes in short-term toxicological studies. The relationship between the pH increase and the cell cleavage needs further investigation.