Control of Epidermal Proliferative Units (EPUs)

A review of the data available on organisation in the differentiated compartment of epidermis has led to the formulation of a hypothesis. This suggests that some degree of control operates within neighbouring groups of epidermal proliferative units (EPUs) determining the input of cells to the highly ordered columns of cornified cells. It seems that this inter-unit control results in groups of units being in "synchrony". It is obvious that some degree of intra-unit control also operates.     

Epidermal and Guard Cell Interactions on Stomatal Aperture in Epidermal Strips and Intact Leaves

Despite the observation first made by von Mohl in 1856, thatepidermal cells greatly influence stomatal aperture, subsequentstudies have failed to pay adequate attention to epidermal cellviability or to quantify the degree of its influence on aperturein epidermal strips and leaf sections. Using Vicia faba stripsand leaf sections we found the following: (i) a non-linear relationshipbetween aperture and guard cell contact with live epidermalcells; (ii) epidermal cell viability on isolated strips hada threshold at about 25 °C; (iii) epidermal strips withdead epidermal cells had wider apertures and lower variabilitythan strips with live cells or intact leaf sections; (iv) afterepidermal cell viability was accounted for, stomatal aperturesshowed no significant differences between isolated strips orstrips removed from leaf sections treated in the same manner;(v) highly variable apertures appeared to be the normal conditionof the intact leaf. Caution should therefore be used in interpretingstomatal behaviour from epidermal strips without first takinginto account mechanical interactions between the guard and surroundingepidermal cells. Vicia faba L, broad bean, epidermal strips, leaf impressions, stomata, guard cells, temperature effects     

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a G_s-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection ¹. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.     

Interactions among the Epidermal Growth Factor-like Modules of Thrombospondin-1

Epidermal growth factor (EGF)-like modules are defined in part by six cysteines joined by disulfides in a 1–3, 2–4, and 5–6 pattern. Thrombospondin-1 (TSP-1) is a multimodular glycoprotein with three EGF-like modules, E1, E2, and E3, arranged in tandem. These modules likely propagate conformational changes between surrounding C-terminal and N-terminal elements of TSP-1 and interact with other extracellular molecules. E1, E2, and their homologs in other TSPs are unique among EGF-like modules in having two residues rather than one between Cys-4 and Cys-5. In addition, E2 has a calcium-binding site and an unusually long loop between Cys-5 and Cys-6. The structure of E1, E2, or E3 expressed alone changed little upon heating as monitored by far-UV CD, whereas more marked changes occurred in E12, E23, and E123 tandem constructs. The individual modules denatured in differential scanning calorimetry experiments only at >85 °C. E12, E23, or E123 tandem constructs, however, had a transition in the range of 44–70 °C. The temperature of the transition was higher when calcium was present and higher with E123 than with E12 or E23. Isothermal titration calorimetry demonstrated K_D values of binding of calcium to E2, E12, E23, or E123 at 25 °C of 11.5, 2.9, 2.2, or 0.3 μm, respectively. Monoclonal antibodies HB8432 and C6.7, which recognize epitopes in E2, bound to E12, E23, or E123 with greater affinity than to E2 alone. These results indicate that interactions among the modules of E123 influence the tertiary structure and calcium binding of E2.Thrombospondins (TSPs)² are multimodule, calcium-binding extracellular glycoproteins with various functions (1). TSP-1, which was the first TSP to be discovered and remains the best characterized, and TSP-2 are trimers. Each subunit is composed of an N-terminal module, oligomerization domain, von Willebrand factor type C module, three properdin or TSP type 1 modules, and the C-terminal signature domain that includes three EGF-like modules (E123), 13 aspartate-rich calcium-binding repeats of the wire module, and a lectin-like module (2–4). The five mammalian TSPs fall into two groups, trimeric (TSP-1 and TSP-2) and pentameric (TSP-3, TSP-4, and TSP-5) (1). All have a signature domain, with the major difference being the presence of four rather than three EGF-like modules in the signature domain of pentameric TSPs.EGF-like modules exist in more than 300 human extracellular proteins and play important roles in biological processes such as blood clotting and cell-cell signaling (5–7). The modules are 30–50 residues long and characterized by six cysteine residues that form three disulfide bonds in the order 1–3, 2–4, and 5–6 (Fig. 1) (6, 7). The backbone structure of the EGF-like modules consists of two submodules, referred to as the major (N-terminal) and minor (C-terminal) submodules (6, 8, 9).Open in a separate windowFIGURE 1.Model of the structure of E123. The model is built based on the crystal structure of EGF modules in the TSP-2 signature domain (Protein Data Bank code 1YO8) using SYBYL 7.0. E1 is shown in red, E2 in pink, and E3 in purple. The cysteines are colored yellow; the backbones of the residues between the fourth and fifth Cys are in blue; Glu-609 recognized by HB8432 and C6.7 is shown in green; and the long loop in E2 between the fifth and sixth Cys is hot pink. Ca²⁺ bound to the binding site on E2 near the interface between E1 and E2 is depicted as a red ball.The crystal structure of the three EGF-like modules of TSP-2 has been solved as part of the TSP-2 signature domain in 2 mm calcium (Ca²⁺) (Fig. 1) (4). All have the 1–3, 2–4, and 5–6 disulfide pattern. There is one Ca²⁺-binding site in the second EGF-like module (E2), located near the interface between the first and second EGF-like modules (E1 and E2) (Fig. 1). There is only one residue between the fourth and fifth cysteines in most EGF-like modules (6). However, E1 and E2 of TSP-1 and TSP-2 and three of the four EGF-like modules (E1, E2, and E2′) of pentameric TSPs have two residues between the fourth and fifth Cys. This difference is potentially important because the N-terminal major submodule of the repeat containing the 1–3 and 2–4 disulfides and the C-terminal submodule with the 5–6 disulfide have the potential to undergo hinge-like motions around the residues between the fourth and fifth Cys (6, 8, 9). Having two rather than one residue between these two Cys increases the potential flexibility. In addition, E2 modules in all five TSPs contain an unusually long loop of 23 residues between the fifth and sixth Cys (Fig. 1). In the TSP-2 signature domain structure, residues from the long loop interact with repeat 12N of the wire module (4). E3, which has one residue between the fourth and fifth Cys, interacts with the wire and the lectin-like module (3, 4). A common polymorphism (N700S) in wire repeat 1C of human TSP-1 influences the stability of the EGF-like modules (10). This finding suggests that the interactions between the EGF-like modules and more C-terminal elements of the signature domain allow conformational changes in the more C-terminal elements to be propagated N-terminally.The EGF-like modules (E123) of TSP-1 denature in differential scanning calorimetry (DSC) with a melting temperature of ∼68 °C in 2 mm Ca²⁺ (10), although most EGF-like modules are stable to heating (7). We have investigated this transition in detail to learn its origins and the influence of Ca²⁺. The results indicate interactions among the modules of E123 that enhance Ca²⁺ binding and influence the tertiary structure of E2.     

Extra-Cutaneous Melanin

Extra-cutaneous melanocytes derive from either the neural crest, the outer wall of the optic cup, or the cranial neural tube. Those of neural crest origin reach most bodily regions, and may give rise to primary melanoma in various tissues. The Kupffer cell produces a form of melanin, but is hardly a melanocyte. Melanocytes of the internal ear may be concerned with secretion of endolymph, trans-epithelial ion transport, and with protection against ototoxic drugs and high-intensity noise damage. There is evidence from albino animals that retinal pigment epithelium determines co-ordinates of the neural retina, and its decussation pattern during development. Neuromelanin derives from Dopamine, and is found in dopaminergic neurons widely distributed throughout the brain-stem and hypothalamus, and which project to the striatum and limbic system. Parkinsonism is due to degeneration of melanin-containing dopaminergic neurons of locus coeruleus and substantia nigra, and MPTP provides an investigative probe for studying the causes of Parkinsonism. Neuromelanin should not be regarded as a waste-product, but as something which can affect the firing properties of neurons with specific functional effect.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.     

Rapid Bleach for Melanin

Complete bleaching of melanin in strongly pigmented specimens embedded in paraffin or polystyrene, and sectioned and mounted on slides, is possible in 1-3 hr at 37 C in a solution of 20 ml of benzyl alcohol, 10 ml of acetone, 5 ml of 10% hydrogen peroxide and 4 drops of a 25% ammonia solution. The bleached tissues are well preserved and tolerate further histochemical treatments. A11 the stains and reactions tested give results identical to or better than those obtained after 24-48 hr oxidation in 10% hydrogen peroxide.     

产黑色素酵母状真菌

黑酵母是可产生黑色素的一类酵母菌总称.它们一般生长缓慢,形态复杂,抗逆性强.本文综述了黑酵母分类鉴定、生态学、抗逆性、致病性和黑色素应用等方面的研究进展.Hortaeawerneckii已成为研究盐胁迫响应蛋白及固醇合成的新的模式生物,Aureobasidiumpullulans也有望成为研究发育调控的模式种类.     

Interaction Between Chemicals and Melanin

Various drugs and other chemicals, such as organic amines, metals, polycyclic aromatic hydrocarbons, etc., are bound to melanin and retained in pigmented tissues for long periods. The physiological significance of the binding is not evident, but it has been suggested that the melanin protects the pigmented cells and adjacent tissues by adsorbing potentially harmful substances, which then are slowly released in nontoxic concentrations. Long-term exposure, on the other hand, may build up high levels of noxious chemicals, stored on the melanin, which ultimately may cause degeneration in the melanin-containing cells, and secondary lesions in surrounding tissues. In the eye, e.g., and in the inner ear, the pigmented cells are located close to the receptor cells, and melanin binding may be an important factor in the development of some ocular and inner ear lesions. In the brain, neuromelanin is present in nerve cells in the extrapyramidal system, and the melanin affinity of certain neurotoxic agents may be involved in the development of parkinsonism, and possibly tardive dyskinesia. In recent years, various carcinogenic compounds have been found to accumulate selectively in the pigment cells of experimental animals, and there are many indications of a connection between the melanin affinity of these agents and the induction of malignant melanoma.     

Melanin Production by Rhizobium Strains

Different Rhizobium and Bradyrhizobium strains were screened for their ability to produce melanin. Pigment producers (Mel⁺) were found among strains of R. leguminosarum biovars viceae, trifolii, and phaseoli, R. meliloti, and R. fredii; none of 19 Bradyrhizobium strains examined gave a positive response. Melanin production and nod genes were plasmid borne in R. leguminosarum biovar trifolii RS24. In R. leguminosarum biovar phaseoli CFN42 and R. meliloti GR015, mel genes were located in the respective symbiotic plasmids. In R. fredii USDA 205, melanin production correlated with the presence of its smallest indigenous plasmid.     

Melanin Biosynthesis in Cryptococcus neoformans

Pigment production by Cryptococcus neoformans is virulence associated. Dopamine- and 3,4-dihydroxyphenylalanine–melanin products were identified after acidic permanganate oxidation, alkaline hydrogen peroxide oxidation, or hydrolysis with hydriodic acid. These data provide direct chemical evidence for the formation of eumelanin polymers by catecholamine oxidation by laccase alone followed by oxidative coupling of dihydroxyindole.     

Melanin production in Alternaria helianthi

Abstract

Most of the plant pathogenic fungi produce a dark phenolic polymer called melanin. The high performance liquid chromatography (HPLC) analysis of the mycelial extract of Alternaria helianthi revealed an accumulation of scytalone and a shunt metabolite 2-hydroxyjuglone which confirms the production of dihydroxynapthalene type of melanin. The growth and melanin of A. helianthi increased when grown in host extract broth at 6.5 pH and a temperature beyond 30°C had an inhibitory effect on the pathogen. The production and type of melanin produced in Alternaria helianthi is reported for the first time.     

Melanin complex of the fungusInonotus obliquus

The fungusInonotus obliquus (Pers.) Pil. synthesized high-molecular-weight phenolic pigments that were assigned to melanins according to their physicochemical properties. It was shown that copper ions (0.008%), pyrocatechol (1.0 mM), and tyrosine (20.0 mM) stimulated melanogenesis. The production of melanin correlated with the synthesis ofo- andp-diphenoloxidases. The fungal melanin had strong antioxidant and genoprotective effects.     

Electron Spin Resonance Studies on Melanin

Electron spin resonance (e.s.r.) observations of squid melanin have been conducted over the temperature range 500°K to 4.2°K, and the effect of various chemical treatments of the melanin upon the e.s.r. spectrum has been studied. The findings have shown that the paramagnetism of this melanin follows the Curie Law from 500°K to 4.2°K, that the spin signal can be eliminated by the addition of Cu⁺⁺ to the melanin, and that the optical and e.s.r. absorptions of melanin are independent since either can be reduced or eliminated without affecting the other. Similar studies on synthetic melanins produced by autoxidation or by enzymatic oxidation of a number of biphenols were carried out. It was found that the e.s.r. signals of these synthetic melanins were strikingly similar (with respect to line width, line shape, and g-value) with those of squid melanin. It is concluded that the unpaired electrons observed are associated with trapped free radicals in the melanin polymer, that the biosynthesis of melanin may involve a free radical mechanism, and that these physical data are in accord with the concept of Nicolaus that melanin is a highly irregular, three-dimensional, polymer.     

Melanin and HIV in sub-Saharan Africa

HIV is common in sub-Saharan Africa. Sexually transmitted bacterial and fungal infections increase the chance of HIV infection. Melanin can prevent the penetration of skin and mucus membranes by microorganisms, and soluble melanin can inhibit HIV replication. We suggest that melanin may reduce the incidence of HIV infection through venereally acquired skin lesions, thus reducing the risk of sero-conversion and slow the progress to AIDS. Indigenous sub-Saharan peoples are highly melanized, but there is pigment variation between populations. We show that skin reflectance, a negative correlate of melanin, is positively associated with adult rate of HIV in sub-Saharan countries. There is no such relationship in populations outside sub-Saharan Africa. We suggest that melanin concentration in black people may correlate with resistance to HIV infection.