Ação da luz visível na oxidação da melanina e no descolorimento do cabelo / Action of the visible on the melanin oxidation and hair discoloration

O dano capilar causado pelo descolorimento oxidativo é muito intenso, sendo que dois fatores são responsáveis por essa ação: primeiro, a ação direta e danosa do oxidante em diversas estruturas capilares e segundo, o dano oxidativo primário facilita o dano causado por outros agentes físicos (luz, temperatura) e químicos (tensoativos), que comumente tem ação nos cabelos. Desenvolver conceitos e tecnologias que possam tornar o oxidante específico para a melanina e por conseguinte efetuando o descolorimento sem causar danos ao fio é extremamente desejável. Neste trabalho buscaremos entender de que forma a luz visível pode aumentar a ação do oxidante sem danificar o fio colateralmente. O objetivo principal deste trabalho é demonstrar que é possível utilizar a luz visível, que é absorvida pela melanina, para tornar esse pigmento mais suscetível ao agente oxidante e desta forma, permitir que o descolorimento seja realizado com concentrações pequenas de oxidante. Também almejamos desenvolver métodos de análises por microscopia ótica de fluorescência e de reflexão para mensurar o dano nas estruturas dos fios processados com oxidante e na presença ou ausência da luz

The capillary damage caused by oxidative discoloration is very intense, and two factors are responsible for this action: first, the direct and harmful action of the oxidant on several capillary structures and second, the primary oxidative damage facilitates the damage caused by other physical agents (light, temperature) and chemicals (surfactants), which commonly have action on the hair. Developing concepts and technologies that can make the oxidant specific to melanin and therefore discoloring without causing damage to the hair is extremely desirable. In this work we will try to understand how visible light can increase the oxidant's action without damaging the wire collaterally. The main objective of this work is to demonstrate that it is possible to use visible light, which is absorbed by melanin, to make this pigment more susceptible to the oxidizing agent and, thus, to allow the discoloration to be carried out with small concentrations of oxidizer. We also aim to develop methods of analysis by optical fluorescence and reflection microscopy to measure the damage to the structures of the threads processed with oxidizer and in the presence or absence of light

Baicalin and berberine ultradeformable vesicles as potential adjuvant in vitiligo therapy.

0.5-1% of the world's population is affected by vitiligo, a disease characterized by a gradual depigmentation of the skin. Baicalin and berberine are natural compounds with beneficial activities, such as antioxidant, anti-inflammatory and proliferative effects. These polyphenols could be useful for the treatment of vitiligo symptoms, and their efficacy can be improved by loading in suitable carriers. The aim of this work was to formulate and characterize baicalin or berberine loaded ultradeformable vesicles, and demonstrate their potential as adjuvants in the treatment of vitiligo. The vesicles were produced using a previously reported simple, scalable method. Their morphology, size distribution, surface charge and entrapment efficiency were assessed. The ability of the vesicles to promote the permeation of the polyphenols was evaluated. The antioxidant and photoprotective effects were investigated in vitro using keratinocytes and fibroblasts. Further, the stimulation of melanin production and tyrosinase activity in melanocytes after treatment with the vesicles were assessed. Ultradeformable vesicles were small in size, homogeneously dispersed, and negatively charged. They were able to incorporate high amounts of baicalin and berberine, and promote their skin permeation. In fact, the polyphenols concentration in the epidermis was higher than 10%, which could be indicative of the formation of a depot in the epidermis. The vesicles showed remarkable antioxidant and photoprotective capabilities, presumably correlated with the stimulation of melanin production and tyrosinase activity. In conclusion, baicalin or berberine ultradeformable vesicles, and particularly their combination, may represent promising nanosystem-based adjuvants for the treatment of vitiligo symptoms.

7-Azaindole Analogues as Bioactive Agents and Recent Results.

Azaindoles have been accepted as important structures having various biological activities in medicinal chemistry in novel drug discovery. Various azaindole derivatives have been used commercially and newer analogues are synthesized continuously. As in literature, azaindole is a very potent moiety, its derivatives displayed a number of biological activities such as kinase inhibitors, cytotoxic agents, anti-angiogenic activity, CRTh2 receptor antagonists, melanin agonists, nicotine agonists, effectiveness in alzheimer disease, cytokinin analogs, Orai inhibitors in asthma and chemokine receptor- 2 (CCR2) antagonists. This review consists of biological activities of various azaindole analogs, reported so far, and their structure activity relations, along with future perspectives in this field.

Dual hypopigmentary effects of punicalagin via the ERK and Akt pathways.

Punicalagin is a phenolic compound with antioxidant properties. However, the effects of punicalagin on melanin synthesis have been poorly evaluated. Therefore, we investigated the effects of punicalagin on melanogenesis in Mel-Ab cells. Punicalagin significantly inhibited melanin synthesis in a dose-dependent manner. In accordance with the melanin content, punicalagin also dose-dependently decreased tyrosinase activity. Punicalagin did not directly inhibit tyrosinase in a cell-free system but did downregulate the expression of microphthalmia-associated transcription factor (MITF) and tyrosinase. Therefore, we examined the effects of punicalagin on melanogenesis-related signaling pathways. Punicalagin induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation but had no effect on ß-catenin level. We measured melanin content and MITF expression in the presence of the ERK pathway inhibitor PD98059 and/or the Akt pathway inhibitor LY294002. Cotreatment with PD98059 and LY294002 almost completely restored punicalagin-induced hypopigmentation. These data indicate that punicalagin inhibits melanin synthesis through ERK and Akt phosphorylation, with subsequent downregulation of MITF and tyrosinase.

Chemical excitation of electrons: A dark path to melanoma.

Sunlight's ultraviolet wavelengths induce cyclobutane pyrimidine dimers (CPDs), which then cause mutations that lead to melanoma or to cancers of skin keratinocytes. In pigmented melanocytes, we found that CPDs arise both instantaneously and for hours after UV exposure ends. Remarkably, the CPDs arising in the dark originate by a novel pathway that resembles bioluminescence but does not end in light: First, UV activates the enzymes nitric oxide synthase (NOS) and NADPH oxidase (NOX), which generate the radicals nitric oxide (NO) and superoxide (O2(-)); these combine to form the powerful oxidant peroxynitrite (ONOO(-)). A fragment of the skin pigment melanin is then oxidized, exciting an electron to an energy level so high that it is rarely seen in biology. This process of chemically exciting electrons, termed "chemiexcitation", is used by fireflies to generate light but it had never been seen in mammalian cells. In melanocytes, the energy transfers radiationlessly to DNA, inducing CPDs. Chemiexcitation is a new source of genome instability, and it calls attention to endogenous mechanisms of genome maintenance that prevent electronic excitation or dissipate the energy of excited states. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin.

Isosakuranetin, a 4'-O-methylated flavonoid, stimulates melanogenesis in B16BL6 murine melanoma cells.

AIMS: The beneficial effects of 4'-O-methylated flavonoids on induction of melanogenesis are well established. Here, we report the effect of isosakuranetin (Iso) on melanogenesis in B16BL6 melanoma cells and an analysis of the signaling pathways involved in this activity. METHODS: B16BL6 melanoma cells were treated with several concentrations of Iso and melanin content was measured. Activation and expression of factors involved in melanogenesis were assessed via western blotting. KEY FINDINGS: Iso (15 and 30µmol/L) strongly stimulated melanogenesis in a dose-dependent manner. Iso increased tyrosinase activity and up-regulated tyrosinase (Tyr), tyrosinase related protein 1 (TRP1), and tyrosinase related protein 2 (TRP2) in a time-dependent manner. Iso decreased B16 cell proliferation at a concentration above 45µmol/L, and had no effect on cell viability as revealed by MTT and trypan blue assays. Iso up-regulated expression of microphthalmia transcription factor (MITF), with a maximum effect after 12h. H89, a specific inhibitor of PKA, showed that MITF up-regulation is mediated through PKA/CREB activation. Furthermore, Iso decreased phosphorylation of MITF at Ser73 after 24h and 48h of exposure, activating MITF and leading to up-regulation of Tyr, TRP1, and TRP2. Iso inhibited phosphorylation and activation of ERK1/2 after 12h, while no significant effects on p38 and JNK phosphorylation were observed. Iso inhibited AKT phosphorylation and led to activation of GSK3ß. SIGNIFICANCE: Iso stimulates melanogenesis in B16 melanoma cells via up-regulation of MITF. Furthermore, Iso-induced inhibition of ERK1/2 and PI3K/AKT signaling pathways activate MITF and subsequent expression of Tyr, TRP1, and TRP2.

Differential modulation of gonadotropin responses to kisspeptin by aminoacidergic, peptidergic, and nitric oxide neurotransmission.

Kisspeptins (Kp), products of the Kiss1 gene, have emerged as essential elements in the control of GnRH neurons and gonadotropic secretion. However, despite considerable progress in the field, to date limited attention has been paid to elucidate the potential interactions of Kp with other neurotransmitters known to centrally regulate the gonadotropic axis. We characterize herein the impact of manipulations of key aminoacidergic (glutamate and GABA), peptidergic (NKB, Dyn, and MCH), and gaseous [nitric oxide (NO)] neurotransmission on gonadotropin responses to Kp-10 in male rats. Blockade of ionotropic glutamate receptors (of the NMDA and non-NMDA type) variably decreased LH responses to Kp-10, whereas activation of both ionotropic and metabotropic receptors, which enhanced LH and FSH release per se, failed to further increase gonadotropin responses to Kp-10. In fact, coactivation of metabotropic receptors attenuated LH and FSH responses to Kp-10. Selective activation of GABA(A) receptors decreased Kp-induced gonadotropin secretion, whereas their blockade elicited robust LH and FSH bursts and protracted responses to Kp-10 when combined with GABA(B) receptor inhibition. Blockade of Dyn signaling (at κ-opioid receptors) enhanced LH responses to Kp-10, whereas activation of Dyn and NKB signaling modestly reduced Kp-induced LH and FSH release. Finally, MCH decreased basal LH secretion and modestly reduced FSH responses to Kp-10, whereas LH responses to Kp-10 were protracted after inhibition of NO synthesis. In summary, we present herein evidence for the putative roles of glutamate, GABA, Dyn, NKB, MCH, and NO in modulating gonadotropic responses to Kp in male rats. Our pharmacological data will help to characterize the central interactions and putative hierarchy of key neuroendocrine pathways involved in the control of the gonadotropic axis.

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.

BACKGROUND: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. AIM: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. METHODS: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. RESULTS: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. CONCLUSION: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

Effects of intracerebroventricular and intra-accumbens melanin-concentrating hormone agonism on food intake and energy expenditure.

The brain melanin-concentrating hormone (MCH) system represents an anabolic system involved in energy balance regulation through influences exerted on the homeostatic and nonhomeostatic controls of food intake and energy expenditure. The present study was designed to further delineate the effect of the MCH system on energy balance regulation by assessing the actions of the MCH receptor 1 (MCHR1) agonism on both food intake and energy expenditure after intracerebroventricular (third ventricle) and intra-nucleus-accumbens-shell (intraNAcSH) injections of a MCHR1 agonist. Total energy expenditure and substrate oxidation were assessed following injections in male Wistar rats using indirect calorimetry. Food intake was also measured. Pair-fed groups were added to evaluate changes in thermogenesis that would occur regardless of the meal size and its thermogenic response. Using such experimental conditions, we were able to demonstrate that acute MCH agonism in the brain, besides its orexigenic effect, induced a noticeable change in the utilization of the main metabolic fuels. In pair-fed animals, MCH significantly reduced lipid oxidation when it was injected in the third ventricle. Such an effect was not observed following the injection of MCH in the NAcSH, where MCH nonetheless strongly stimulated appetite. The present results further delineate the influence of MCH on energy expenditure and substrate oxidation while confirming the key role of the NAcSH in the effects of the MCH system on food intake.

Cannabinoids excite hypothalamic melanin-concentrating hormone but inhibit hypocretin/orexin neurons: implications for cannabinoid actions on food intake and cognitive arousal.

Cannabinoids modulate energy homeostasis and decrease cognitive arousal, possibly by acting on hypothalamic neurons including those that synthesize melanin-concentrating hormone (MCH) or hypocretin/orexin. Using patch-clamp recordings, we compared the actions of cannabinoid agonists and antagonists on identified MCH or hypocretin neurons in green fluorescent protein-expressing transgenic mice. The cannabinoid type-1 receptor (CB1R) agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpho linylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate (WIN55,212,2) depolarized MCH cells and increased spike frequency; in contrast, WIN55,212,2 hyperpolarized and reduced spontaneous firing of the neighboring hypocretin cells, both results consistent with reduced activity seen with intracerebral cannabinoid infusions. These effects were prevented by AM251 [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide], a CB1R antagonist, and by tetrodotoxin, suggesting no postsynaptic effect on either neuron type. In MCH cells, depolarizing WIN55,212,2 actions were abolished by the GABA(A) receptor antagonist bicuculline, suggesting that the CB1R-mediated depolarization was attributable to reduced synaptic GABA release. WIN55,212,2 decreased spontaneous IPSCs, reduced the frequency but not amplitude of miniature IPSCs, and reduced electrically evoked synaptic currents in MCH cells. Glutamate microdrop experiments suggest that WIN55,212,2 acted on axons arising from lateral hypothalamus local inhibitory cells that innervate MCH neurons. In hypocretin neurons, the reduced spike frequency induced by WIN55,212,2 was attributable to presynaptic attenuation of glutamate release; CB1R agonists depressed spontaneous and evoked glutamatergic currents and reduced the frequency of miniature EPSCs. Cannabinoid actions on hypocretin neurons were abolished by ionotropic glutamate receptor antagonists. Together, these results show that cannabinoids have opposite effects on MCH and hypocretin neurons. These opposing actions could help explain the increase in feeding and reduction in arousal induced by cannabinoids.

PBAN selective antagonists: inhibition of PBAN induced cuticular melanization and sex pheromone biosynthesis in moths.

A D-Phe scan (sequential D-Phe replacement) library of linear peptides, synthesized on the basis of a slightly modified active sequence of PBAN (YFSPRL-amide) was employed to detect potential inhibitors of cuticular melanization in Spodoptera littoralis larvae and to compare their stimulatory and inhibitory melanization activity with their pheromonotropic agonistic and antagonistic activities. A quantitative melanotropic assay was used to monitor the extent of cuticular melanization elicited by Hez-PBAN1-33NH2 in S. littoralis larvae in the presence and absence of the D-Phe peptides. The data revealed the presence of two partial melanotropic antagonists, and disclosed the presence of selective pure melanotropic agonists and pure pheromonotropic antagonists indicating differences in the inhibitory and stimulatory patterns of the library with respect to both activities. The differences between the pheromonotropic and melanotropic inhibitory patterns of the peptides hints at the possibility that sex pheromone biosynthesis in the pheromone gland of Heliothis peltigera females and induction of cuticular melanization in S. littoralis may be mediated by different receptors (that may result either from presence of different receptor sub-types or may reflect species differences in receptor structure and/or properties) despite the fact that they are induced by the same peptide (PBAN1-33NH2).

Synthesis and biological evaluation in vitro of a selective, high potency peptide agonist of human melanin-concentrating hormone action at human melanin-concentrating hormone receptor 1.

Human melanin-concentrating hormone (hMCH) is a nonselective natural ligand for the human melanin-concentrating hormone receptors: hMCH-1R and hMCH-2R. Similarly, the smaller peptide encompassing the disulfide ring and Arg(6) of hMCH, Ac-Arg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Gly(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), Ac-hMCH(6-16)-NH(2), binds to and activates equally well both human MCH receptors present in the brain. To separate the physiological functions of hMCH-1R from those of hMCH-2R, new potent and hMCH-1R selective agonists are necessary. In the present study, analogs of Ac-hMCH(6-16)-NH(2) were prepared and tested in binding and functional assays on cells expressing the MCH receptors. In these peptides, Arg in position 6 was replaced with various d-amino acids and/or Gly in position 10 was substituted with various L-amino acids. Several of the new compounds turned out to be potent agonists at hMCH-1R with improved selectivity over hMCH-2R. For example, peptide 26 with d-Arg in place of L-Arg in position 6 and Asn in place of Gly in position 10, Ac-dArg(6)-cyclo(S-S)(Cys(7)-Met(8)-Leu(9)-Asn(10)-Arg(11)-Val(12)-Tyr(13)-Arg(14)-Pro(15)-Cys(16))-NH(2), was a potent hMCH-1R agonist (IC(50) = 0.5 nm, EC(50) = 47 nm) with more than 200-fold selectivity with respect to hMCH-2R. Apparently, these structural changes in positions 6 and 10 results in peptide conformations that allow for efficient interactions with hMCH-1R but are unfavorable for molecular recognition at hMCH-2R.

Visualizing differences in ligand-induced beta-arrestin-GFP interactions and trafficking between three recently characterized G protein-coupled receptors.

beta-Arrestin 1-GFP or beta-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melanin-concentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both beta-arrestin 1-GFP and beta-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for beta-arrestin 2-GFP. beta-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of beta-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and trafficking of TAMRA-(5- and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of beta-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of beta-arrestin 1-GFP from intracellular vesicles. For the melanin-concentrating hormone receptor the bulk of translocated beta-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of beta-arrestin-GFP interactions and trafficking for three G protein-coupled receptors for which the natural ligands have only recently been identified and which were thus previously considered as orphan receptors.