Analysis of plasmid DNA damage induced by melanin with capillary electrophoresis

Dilute linear poly(N-isopropylacrylamide) (PNIPAM) in Tris-Mes-EDTA (TME) buffer was used as sieving matrix for capillary electrophoresis (CE) of plasmid DNA and plasmid topological isomers induced by melanin in uncoated capillary. At the optimized condition of 0.1% (w/v) PNIPAM in TME buffer, base line separation of the plasmid DNA ladder (2-12 kbp) was achieved within 15 min. Three positive clones with inserts of 468, 1147 and 1566 bp can be distinguished from the plasmid pUC 18 vector within 13 min. The migration order of the plasmid topological isomers in the dynamic coating matrix was confirmed by the enzymatically prepared and UV-induced plasmids. The covalently closed circular form appeared firstly, followed by the linear plasmid form and then the open circular form. The effect of bacterial melanin obtained from Pseudomonas maltophilia AT18 on plasmid pUC 18 was investigated by CE in uncoated capillary in vitro. Plasmid pUC 18 incubated with either melanin or copper ions alone sustained little DNA damage. The combination of melanin with Cu(II) can cause the plasmid pUC 18 conformational changes from covalently closed circular form to open form. Understanding the damage effect of melanin with copper ions on DNA would be important for the melanin-related application, such as photoprotective antioxidant in protecting the skin from cancer, pathophysiology research in clinic. The investigation of melanin induced plasmid conformational changes by CE in uncoated capillary also revealed that the application of the dynamic coating matrix could be extended to the study of plasmid conformational changes in other plasmid-based biological technologies.     

Whitening activity of luteolin related to the inhibition of cAMP pathway in α-MSH-stimulated B16 melanoma cells

To examine the possibility of luteolin as a whitening agent, we measured antioxidant activity using DPPH assay, NBT/XO assay and intracellular ROS scavengning assay and depigmenting activity using tyrosinase assay, alpha-MSH-induced melanin production in B-16 cells. Luteolin showed dose-dependent anti-oxidant activity in DPPH, NBT/XO and intracellular ROS assay. Also, luteolin directly inhibited xanthine oxidase activity in a dose-dependent manner. Although luteolin did not directly inhibit tyrosinase activity, it dose-dependently inhibited both tyrosinase activity and melanin production in B16 melanoma cells stimulated by 1 muM alpha-MSH. Luteolin dose-dependently inhibited cAMP levels in B16 melanoma cells stimulated by 1 muM alpha-MSH and 1 muM forskolin, which suggest that luteolin directly inhibits adenyl cyclase in B16 melanoma cells. Therefore, these results suggest that whitening activity of luteolin may be due to the inhibition of adenyl cyclase involved in the signal pathway of alpha-MSH in B16 melanoma cells.     

Investigation of the feeding effects of melanin concentrating hormone on food intake — action independent of galanin and the melanocortin receptors

Melanin concentrating hormone (MCH) is recognised as a hypothalamic appetite stimulant. The mechanism of action of MCH is undetermined largely due to lack of identification of hypothalamic MCH receptors. We designed in vivo and in vitro studies to further characterise the feeding effects of MCH in the rat. MCH was injected directly into the paraventricular nucleus (PVN) at the beginning of the light phase. PVN MCH (0.5 microg) produced an increase in 2 h food intake of 272+/-60% vs. saline control (0.7+/-0.2 g), p<0.05. The time course of the effect of intracerebroventricular (i.c.v.) administration of 5 microg MCH on food intake was investigated. An increase in feeding was observed within 15 min from the time of injection and was not sustained beyond half an hour following administration. To investigate a possible interaction with galanin, 5 microg of MCH was injected i.c.v. with or without 10 microg of galanin. The two peptides together increased 1 h feeding above that of either peptide alone, 768+/-62% (compared with the saline group, 0.47+/-0.2 g), p<0.05 vs. 585+/-36%, galanin alone and 317+/-72%, MCH alone. Finally, to investigate if MCH bound to the brain melanocortin receptors, receptor autoradiography was performed on rat brain sections with the stable analogue of alpha MSH, [125I] Nle(4), D-Phe(7)-alphaMSH and unlabeled MCH. MCH did not compete with [125I] Nle(4), D-Phe(7)-alphaMSH binding. Results demonstrate that MCH stimulates feeding via the PVN, has a short onset and duration of action and activates feeding by mechanisms independent to galanin and the melanocortin receptors.     

Mice lacking Melanin Concentrating Hormone 1 receptor are resistant to seizures

The Melanin Concentrating Hormone (MCH) system is widely expressed throughout the central nervous system and regulates a variety of physiological functions. It has been reported that acute central administration of MCH inhibits pentylenetetrazol (PTZ)-induced seizures in rats. In the present study MCH₁ receptor knockout mice (MCH₁R-KO) were used to investigate the role of MCH signaling in modulating seizure susceptibility. Seizure behaviors were compared between MCH₁R-KO and wild type (MCH₁R-WT) mice following administration of the convulsant compounds PTZ or pilocarpine. PTZ injection induced clonic seizures in MCH₁R-WT mice but failed to induce them in MCH₁R-KO mice. More than twice as many injections of intermittently administered low dose PTZ were required to induce clonic seizures in MCH₁R-KO mice than in MCH₁R-WT mice. Following pilocarpine injection, MCH₁R-WT mice experienced clonic seizures and most had tonic seizures and entered status epilepticus, while all MCH₁R-KO mice were completely resistant to these effects. MCH₁R-KO mice were also observed to be strongly protected from the development of PTZ kindling. Genetic deletion of MCH₁R conferred resistance to all seizure models tested in this study. The data indicate that the MCH system is involved in the regulation of PTZ and pilocarpine seizure threshold.     

内服黑素再生液联合窄谱中波紫外线治疗白癜风护理体会

总结内服黑素再生液联合窄谱中波紫外线治疗白癜风的疗效观察,并给予病人有效的术前、术中、术后护理,帮助病人解除思想顾虑,进行心理、饮食及术后恢复的指导,以增加治愈率。     

Non-toxic melanin production inhibitors from Garcinia livingstonei (Clusiaceae)

Ethnopharmacological relevance

The stem bark of Garcinia livingstonei is used traditionally as a skin lightening agent.

Aim of the study

To isolate and identify compounds responsible for the observed skin lightening activity of Garcinia livingstonei and to evaluate their cytotoxicity.

Materials and methods

Constituents of the stem bark and fruits of Garcinia livingstonei were isolated using chromatographic techniques and structures were determined using 1D and 2D NMR and MS analysis. MeWo cells were used to evaluate the cytotoxicity and impact on melanin levels of extracts and compounds isolated, in vitro.

Results

Twelve known compounds, morelloflavone (1), morelloflavone-7″-sulphate (2), guttiferone A (3), sargaol (4), isojacareubin (5), 6-deoxyisojacareubin (6) and in addition to the common triterpenoids, betulin, betulin aldehyde, lupeol, lupenone, euphol and stigmasterol were isolated in this investigation. Morelloflavone, morelloflavone-7″-sulphate and sargaol, were found to be considerably less cytotoxic and more effective as skin lightening agents than hydroquinone.

Conclusions

A range of compounds was isolated from the stem bark and fruit of Garcinia livingstonei. Although the bark extract contained the cytotoxic guttiferone A, it was found to be less toxic than hydroquinone, and morelloflavone, the 7″-sulphate derivative and sargaol show potential for development as depigmentation/skin lightening agents.     

Facial-Skin Hyperpigmentation： Histological and Computer Geometric Assessment

Hyper-pigmentation is a common, acquired dermatological skin-disorder manifesting and identifiable as irregular brown or greyish-brown facial discolouration, and sometimes referred to as melanosis, melasma or hypermelanosis. Purpose and Objective： To identify the site of melanin deposition in skin-layers regarding facial hyper-pigmentation, based on a histological study of full-thickness skin-facial biopsies in aged Caucasian-cadavers. Hypothesis： Recalcitrant hyper-pigmentation, is chiefly characterised by hyper-melanosis restricted to the dermal-layer of the integument. Method： The histological features of facial hyper-pigmentation and solar-lentigenes were evaluated in a pilot-study of 5-randomly selected Caucasian-cadavers with pigmentation （15 facial biopsies）, ranging in age between 75 and 102 years （mean 77-years）. Selection-criteria included, both genders, age 〉 75, focal and confluent hyper-pigmented lesions, involving sun-exposed areas of skin （centrifacial, scalp, malar, mandibular and cervical）. Study groups included （Grp-1： Control skin-histology in otherwise normal aged, human-cadavers; Grp-2： Histology of pigmented facial skin-lesions in man; Grp-3： Comparative histological skin-controls in non-human primates）. No obvious hepatic disease was evident in the cohort studied. Twenty-five histological controls were obtained from non-pigmented areas. Histological evaluation of full-thickness skin-biopsies （including the lesion, edge and peri-]esion skin）, was under a Leitz~-light-microscope, and staining included H＆E, Masson-trichrome, Masson-fontana, Alcian-blue and Verhoef technology. Histological-scoring used was on histological deposition of melanin in skin-layers： epidermal, dermal, mixed, and indeterminate melanin-deposition （score 1-4）. Controls included cadaveric skin-biopsies of human races of colour and non-human primate, Cercopethicus Aethiops （latter is known to have predominantly dermal-melanin deposition）. Pigmented and non-pigmented areas were compared in both species. Results： The majority of clinically visible individual and confluent areas of hyper-pigmentation studied were maeroscopically present on the forehead, frontal scalp in hair-receded cadavers, molar and temporal zones. Histologically, documented features of age-related changes without pigment were present in almost all the embalmed cadaver-skins, with a melanin-score of 1. Computer enhanced skin geometry and biometrics confirmed the presence of an aged-skin, pigmentation and features of solar damage. The human embalmed-tissue was well preserved and minimal autolytic changes were present. Special stains of full-thickness biopsies （Masson-Fontana）, showed that melanin in the subhuman-primate is lodged in the deep dermis （reticular dermis）, within the extra-cellular matrix （ECM） and superficial to the hypodermal adipose-tissue （melanin-score 3）. Fifteen pigmented lesions studied in five （5） aged-cadavers （forehead, molar and mandibular areas） all showed predominantly epidermal-deposition of melanin in the basal, suprabasal and stratum corneum with tiny focal areas of dermal melanosis in single-cell macrophages in the papillary-dermis but not reticular-dermis （melanin-score 2）. A melanin-deposition localization ratio of epidermis to dermis was approximately 98 to 2% in cadavers with hyper-pigmentation. Conclusion： The skin-strata localization of the melanin with regards hyper-pigmentation of the face and forehead in this aged, human adult Caucasian, cadaveric-study, was predominantly in the epidermis and sparse in the papillary dermis.