β-lapachone accelerates the recovery of burn-wound skin

β-lapachone is a quinone of lapachol extracted from the bark of lapacho tree. Recent findings demonstrated that punched skin wounds of mice healed faster with β-lapachone treatment. The present study investigates the effects of β-lapachone on burn-wound skin of C57BL/6 mice injured by a 100 °C iron stick. Our results indicated that wounds treated with β-lapachone recovered faster than those treated with control ointment containing no β-lapachone. On the third day after burning, the area of β-lapachone treated-wound was 30% smaller than wound treated with control ointment. H&E and immunohistochemistry staining showed that burn-wound skin treated with ointment containing β-lapachone healed faster in its epidermis, dermis, and underlying connective tissues with more macrophages appeared than those treated with control ointment alone. RAW264.7 cell, a macrophage-like cell line derived from BALB/C mice, was used as a model for scrutinizing the effect of β-lapachone on macrophages. We found that the proliferation and the secretion of EGF and VEGF by macrophages were higher in cultures treated with β-lapachone and that ?-lapachone can also increase the release of EGF with TNF-α pretreatment. We conclude that β-lapachone plays an important role in accelerating burn wound healing, and that β-lapachone not only can raise the proliferation of macrophages but also increase the release of VEGF from macrophages.     

Alkaloids of anuran skin: antimicrobial function?

A variety of alkaloids, most of which occur or are structurally related to alkaloids that occur in skin glands of dendrobatid poison frogs, were assayed for antimicrobial activity against the Gram-positive bacterium Bacillus subtilis, the Gram-negative bacterium Escherichia coli and the fungus Candida albicans. Certain pyrrolidines, piperidines and decahydroquinolines, perhydro-histrionicotoxin, and a synthetic pumiliotoxin were active against B. subtilis. Only 2-n-nonylpiperidine was active against E. coli. One pyrrolidine, two piperidines, two decahydroquinolines, and the synthetic pumiliotoxin were active against the fungus C. albicans. The results suggest that certain of the skin alkaloids of poison frogs, in addition to being noxious to predators, may also benefit the frog through protection against skin infections.     

Perifosine sensitizes UVB-induced apoptosis in skin cells: New implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.     

Purification and characterization of cholecystokinin from the skin of salamander Tylototriton verrucosus

As a group of intestinal hormones and neurotransmitters, cholecystokinins(CCKs) regulate and affect pancreatic enzyme secretion, gastrointestinal motility, pain hypersensitivity, digestion and satiety, and generally contain a DYMGWMDFG sequence at the C-terminus. Many CCKs have been reported in mammals. However, only a few have been reported in amphibians, such as Hyla nigrovittata, Xenopus laevis, and Rana catesbeiana, with none reported in urodele amphibians like newts and salamanders. Here, a CCK called CCK-TV was identified and characterized from the skin of the salamander Tylototriton verrucosus. This CCK contained an amino acid sequence of DYMGWMDF-NH2 as seen in other CCKs. A c DNA encoding the CCK precursor containing 129 amino acid residues was cloned from the c DNA library of T. verrucosus skin. The CCK-TV had the potential to induce the contraction of smooth muscle strips isolated from porcine gallbladder, eliciting contraction at a concentration of 5.0x10-11 mol/L and inducing maximal contraction at a concentration of 2.0x10-6 mol/L. The EC50 was 13.6 nmol/L. To the best of our knowledge, this is the first report to identify the presence of a CCK in an urodele amphibian.     

Transport of sodium and chloride across earthworm skin in vitro

We present a new invertebrate model for the study of epithelial sodium transport in tight epithelia, the earthworm integument. Dissected segments of earthworm integument were mounted in modified Ussing chambers and perfused with either pond water (PW) or earthworm ringer solution (ERS) on the apical side. In order to investigate ion transport under near-in vivo physiological conditions, measurements were performed under current-clamp conditions by monitoring the transepithelial potential (V _T), as well as the transepithelial resistance (R _T). These were recorded continuously and the virtual short circuit current (I _SC) was calculated. The integument has a high transepithelial resistance (R _T=9,037±502 Ω cm² for PW, n=24, and 11,055±1,320 Ω cm² for ERS, n=32). V _T was −3.7±2.2 mV for PW (n=24) and −1.5±1.0 mV for ERS (n=32), and I _SC was −0.57±0.30 μA/cm² for PW (n=24) and −0.44±0.24 μA/cm² for ERS (n=32). Only under PW, but not under ERS conditions, was there a pronounced inhibition of I _SC by low doses of amiloride or its analogues phenamil and benzamil. The resistance of the paracellular pathway was found to be very high. The terrestrial oligochaete Lumbricus seems especially adapted to the environmental conditions because it has an ultra-tight integument and a very fast up- and down-regulation of apical Na⁺ channels.     

Demonstration of β-glucan receptors in the skin of aquatic mammals—a preliminary report

Using immunohistochemistry, the study clearly demonstrates three important β-glucan receptors (Ficolin/P35, MBL, Dectin-1; members of the lectin-complement pathway of innate immunity) in the integument of six marine and freshwater aquatic mammals (Northern fur seal, Common seal, Walrus, Coypu, Capybara, Otter), but only weakly in two dolphin species. Most of the non-dolphin mammals exhibited strong reactions, especially with regard to the skin glands (tubular apocrine glands, sebaceous glands), for L-Ficolin/P35 and MBL. Distinct reaction staining could also be observed in the epidermis and the outer epithelial sheath of primary hair follicles. Positive Dectin-1 staining was limited to secretory cells of the apocrine tubular glands, and to peripheral and central cells of sebaceous glands of the seals. The Capybara was the only animal to show a clear Dectin reaction in the epidermis (stratum granulosum). The findings are discussed with regard to the constant and high microbial challenge of the skin in the aquatic medium, and variations in hair density of the animals.     

Characterization of β-D-xyloside-initiated glycosaminoglycan synthesized by human skin fibroblasts in the presence of tunicamycin

Human skin fibroblasts were incubated with a fluorogenic xyloside, 4-methylumbelliferyl--D-xyloside (Xyl-MU), in the presence or absence of tunicamycin. The xyloside-initiated glycosaminoglycans (GAG-MUs) were isolated from the culture medium, and their structures characterized. When the cells were incubated with Xyl-MU in the presence of 0.2 g ml–1 tunicamycin, the synthesis of GAG-MU was increased about three fold, compared with the control value in the absence of tunicamycin (cells exposed to Xyl-MU alone). The structures of GAG-MUs synthesized in the presence or absence of tunicamycin were compared by HPLC analysis using gel-filtration and ion-exchange columns, enzymatic digestion, and unsaturated disaccharide composition analysis. The data indicated that cells incubated with tunicamycin produced more undersulfated and shorter GAG-MUs than cells without tynicamycin. These results suggest that tunicamycin inhibits the elongation and sulfation of glycosaminoglycan (GAG) chains and that, as a result, GAG-MUs with shorter chains and undersulfated residues, but possessing a large number of GAG chains, are synthesized in the presence of tunicamycin.     

The Göttingen minipig for assessment of retinoid efficacy in the skin: comparison of results from topically treated animals with results from organ-cultured skin

Göttingen minipigs were treated topically for 6 d with a novel retinoid (MDI 301) at concentrations ranging from 0.3% to 30% in cream vehicle. Treatment of the minipigs did not adversely affect their health (hematological and necropsy parameters) or produce changes in the skin suggestive of retinoid-induced skin irritation. After killing the animals, skin samples from each treatment site were excised and maintained in organ culture for 6 d. In addition, untreated skin was also maintained in organ culture and treated with MDI 301 (0.1–5 μg/ml). After 3 d, the culture supernatants were collected and analyzed for levels of collagen type I and for matrix metalloproteinases (MMPs). Both skin samples treated in vivo and skin samples exposed to MDI 301 in culture demonstrated increased collagen production. Only slight changes in levels of MMP-2 (gelatinase A) or MMP-9 (gelatinase B) were seen. After 6 d, the organ-cultured skin was fixed in formalin and prepared for histology. The organ-cultured skin was compared to skin that was fixed at killing after in vivo treatment. Epidermal hyperplasia was quantified at various MDI 301 concentrations. In vivo and in vitro treatments showed similar results—although the thickness was not substantially changed on average, there were focal areas of hyperplasia at higher retinoid concentrations. Taken together, these data suggest that MDI 301 enhances collagen production in minipig skin, without irritation. Furthermore, these studies suggest that minipig skin exposed to the retinoid in organ culture is equally predictive as topically treated skin. The in vitro organ culture approach may provide a cost-effective alternative model to that of the intact animal for skin retinoid testing.     

Purification to homogeneity of the human skin chymotryptic proteinase “chymase”

The chymotrypic proteinase “chymase” has been purified to apparent homogeneity from human skin. Our procedure differs from previously published partial purifications in that it does not involve affinity chromatography, most of the steps are carried out in 2 m KCl which stabilizes the enzyme, detergent is used to protect the enzyme in low-ionic-strength media, and troublesome concentration steps are avoided by using very small columns of high-capacity exchangers. The high-salt skin extract is applied successively to columns of hydroxyapatite, copper chelate Sepharose, and Sephadex G-100 in 2 m KCl. After dialysis against a zwitterionic detergent, the enzyme is adsorbed onto a 0.4-ml column of CM-Sepharose. An alkaline wash removes the remaining contaminants from the highly cationic enzyme, which is then eluted with 1 m KCl in a final volume of 2 ml. Sodium dodecyl sulfate electrophoresis reveals a single diffuse band of M_r 30,000. Recoveries range from 20 to 40% with yields of 0.2 to 0.4 mg of enzyme from 200 g of skin. Specific activities vary from 600 to 1400 units/mg for the hydrolysis of acetyltyrosine ethyl ester.     

Telocytes in human skin – are they involved in skin regeneration?

Telocytes (TCs), a particular interstitial cell type, have been recently described in a wide variety of mammalian organs ( www.telocytes.com ). The TCs are identified morphologically by a small cell body and extremely long (tens to hundreds of μm), thin prolongations (less than 100 nm in diameter, below the resolving power of light microscopy) called telopodes. Here, we demonstrated with electron microscopy and immunofluorescence that TCs were present in human dermis. In particular, TCs were found in the reticular dermis, around blood vessels, in the perifollicular sheath, outside the glassy membrane and surrounding sebaceous glands, arrector pili muscles and both the secretory and excretory portions of eccrine sweat glands. Immunofluorescence screening and laser scanning confocal microscopy showed two subpopulations of dermal TCs; one expressed c‐kit/CD117 and the other was positive for CD34. Both subpopulations were also positive for vimentin. The TCs were connected to each other by homocellular junctions, and they formed an interstitial 3D network. We also found TCs adjoined to stem cells in the bulge region of hair follicles. Moreover, TCs established atypical heterocellular junctions with stem cells (clusters of undifferentiated cells). Given the frequency of allergic skin pathologies, we would like to emphasize the finding that close, planar junctions were frequently observed between TCs and mast cells. In conclusion, based on TC distribution and intercellular connections, our results suggested that TCs might be involved in skin homeostasis, skin remodelling, skin regeneration and skin repair.     

Apical Na+ permeability of frog skin during serosal Cl− replacement

Summary Gluconate substitution for serosal Cl^– reduces the transepithelial short-circuit current (I _sc) and depolarizes shortcircuited frog skins. These effects could result either from inhibition of basolateral K⁺ conductance, or from two actions to inhibit both apical Na⁺ permeability (P _Na ^ap ) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na⁺ concentration (C _Na ^c ) andP _Na ^ap have been monitored by measuring the currentvoltage relationship for apical Na⁺ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates ofP _Na ^ap ) and CNa/c were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl^– replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition ofI _sc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations.P _Na ^ap was either unchanged or increased, and CNa/c either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl^– replacement inhibitsP _Na ^ap and Na, K-pump activity. Gluconate substutition likely reduces cell volume, triggering inhibition of the basolateral K⁺ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C Macknight (J. Membrane Biol. 83:119–137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na⁺ entry. The volume-conductance coupling serves to conserve volume by reducing K⁺ solute loss. Its molecular basis remains to be identified.     

Properties of α-l-iduronidase in cultured skin fibroblasts from α-l-iduronidase-deficient patients

Summary On DEAE cellulose column chromatography, -l-iduronidase in cultured skin fibroblasts was resolved into two distinct components, forms A and B. They had similar K_m values for 4-methylumbelliferyl--l-iduronide, but differed in pH optima and thermal stability. Form B was more heat-stable than form A.Residual -l-iduronidase activity in Hurler fibroblasts was heat-stable, while that in Scheie fibroblasts was heat-labile, and moreover, that in Hurler-Scheie compound fibroblasts lay intermediate between Hurler and Scheie syndromes. These findings demonstrated that Hurler syndrome, Scheie syndrome and Hurler-Scheie compound were enzymatically distinguishable.     

Probiotics for the skin: a new area of potential application?

AIMS: The current study aimed at assessing, in vitro, the potential use of probiotics for the skin. METHODS AND RESULTS: Propionibacteria were chosen as potential probiotics as they are members of the normal cutaneous microbiota. Dairy strains were chosen because of their documented safe use. Production of anti-microbial substances was assessed, against selected skin pathogens. Only production of organic acids was detected. Two of the tested strains were found to exhibit high adhesion to human keratin, in vitro. Despite this high adhesion, no inhibition of skin pathogen adhesion to human keratin was observed. CONCLUSIONS: The current strains assessed may not be optimal for use as skin probiotics. However, the results of the study show that the methodology works for investigating this kind of application. SIGNIFICANCE AND IMPACT OF THE STUDY: Methods for selecting probiotics for potential application on the skin are presented.