Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin-1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin-1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision.

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six-grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Bis[1,2-bis-(meth-oxy-carbon-yl)ethene-1,2-dithiol-ato-κ(2) S,S']bis-(η(5)-penta-methyl-cyclo-penta-dien-yl)tetra-µ3-sulfido-tetra-iron(4 Fe-Fe) hexa-fluoridophosphate.

The asymmetric unit of the title compound, [Fe4(C6H6O4S2)2(C10H15)2S4]PF6, contains two different complex cations and two PF6 (-) anions. The two complex cations have similar conformations with the butterfly-like Fe4S4 core surrounded by two penta-methyl-cyclo-penta-dienyl ligands and the S atoms of two dithiol-ate ligands. In each Fe4S4 core, there are four short Fe-Fe and two long Fe⋯Fe contacts, suggesting bonding and non-bonding inter-actions, respectively. The Fe-S distances range from 2.1287 (13) to 2.2706 (16) Šfor one and from 2.1233 (13) to 2.2650 (16) Šfor the other Fe4S4 core. The Fe-S distances involving the dithiol-ate ligands are in a more narrow range [2.1764 (16)-2.1874 (13) Šfor one and 2.1743 (14)-2.1779 (16) Šfor the other cation]. There are no significant inter-actions between cations and anions.

Bis[1,2-bis-(eth-oxy-carbon-yl)ethene-1,2-dithiol-ato-κ(2) S,S']bis-(η(5)-penta-methyl-cyclo-penta-dien-yl)tetra-µ3-sulfido-diiron(IV)diiron(III)(3 Fe-Fe).

The title compound, [Fe4(C10H15)2(C8H10O4S2)2S4], contains a twisted Fe4S4 cubane-like core. A twofold rotation axis passes through the Fe4S4 core, completing the coordination of the four Fe atoms with two penta-methyl-cyclo-penta-dienyl ligands and two chelating dithiol-ate ligands. There are three short Fe-Fe and three long Fe⋯Fe contacts in the Fe4S4 core, suggesting bonding and non-bonding inter-actions, respectively. The Fe-S bonds in the Fe4S4 core range from 2.1523 (5) to 2.2667 (6) Šand are somewhat longer than the Fe-S bonds involving the dithiol-ate ligand.

Tetra-µ3-selen-ido-1:2:3κ3Se;1:2:4κ3Se;1:3:4κ3Se;2:3:4κ3Se-tetrakis-[(η5-methyl-cyclo-penta-dien-yl)molybdenum(III)](6 Mo-Mo).

The title cluster compound, [Mo4(η5-C5H4Me)4(µ3-Se)4], was synthesized from the reaction of [Mo(η5-C5H4Me)(CO)3]2 with grey selenium in refluxing xylene solution under a nitro-gen atmosphere. The complete cluster is generated by a crystallographic twofold axis and contains an Mo4Se4 cubane-like core surrounded by four η5-methylcyclo-pentadienyl ligands. In the core, the four molybdenum atoms are connected to each other to form a tetra-hedron, with a selenium atom capping each face. The Mo-Mo bond lengths vary from 2.9857 (5) to 3.0083 (3) Šand the Mo-Se separations range from 2.4633 (4) to 2.4693 (5) Å.

Inhibitory effect of the extract of rhizome of Curcuma longa L in gelatinase activity and its effect on human skin.

Exposure to UV radiation to human skin up-regulates the synthesis of matrix metalloproteinase (MMP) family. Gelatinases are member of MMPs which have been suggested to play an important role in photoaging such as wrinkle formation. To inhibit gelatinase activity is regarded to be very important to keep healthy skin and to protect wrinkle formation. On the other hand, anti-photoaging agents are expected to be derived from natural resources, especially plants. Plant extracts having gelatinase-inhibitory effect that might be used as safe anti-photoaging ingredient were widely screened. An extract of rhizomes of Curcuma longa L. showed inhibitory effect of gelatinase activity. Curcuminoids and slight amount of compound, 6,11-dihydroxy-3-(4-hydroxy-3-mthoxyphenethyl)-7-[(E)-4-(4-hydroxy-3-methoxyphenyl)-2-oxo-3-butenyl]-10-methoxy-2-oxabicyclo[6.3.1.]dodeca-1(11),8(12),9-trien-5-yl (E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoate (curcuminoid D) were isolated as the gelatinase-inhibitory components from methanol extract of rhizomes. The structure of curcuminoid D was determined by means of spectral data including 1H- and 13C-NMR, and IR. Curcumin exerted the enhancing effect on deposition of basement membrane component at dermal-epidermal junction in skin equivalent model. Topical application of cream containing turmeric extract significantly improved facial skin elasticity and decreased the number of gelatinase-positive stratum corneum clusters in human facial skins. These results indicated that turmeric is an effective ingredient to improve skin condition and to prevent skin from photoaging by suppressing activation of gelatinase chronically caused by UV.

Non-invasive study of gelatinases in sun-exposed and unexposed healthy human skin based on measurements in stratum corneum.

Gelatinases, which belong to the family of matrix metalloproteinases, degrade various components of skin, and may be involved in photoaging, since they are upregulated by low-dose UV exposure to the skin. However, their behavior in healthy human skin is still unclear. In the present study, gelatinases was specifically detected in stratum corneum (SC) of skin from sun-exposed sites, including the face, in healthy humans, but not in SC of skin from unexposed sites. Following experimental UVB irradiation of the abdomen in volunteers, gelatinases were detected in tape-stripped SC from the site for several weeks, and subsequently disappeared. The appearance of gelatinase in SC after a lag time consistent with SC turnover is considered to reflect upregulation of gelatinase expression in keratinocytes in response to UVB-exposure of the skin. A survey of gelatinases in facial SC samples collected by tape-stripping from the cheeks of 100 healthy women revealed that the enzyme was present in 90% of subjects. These results, taken together, suggest that gelatinase is constantly upregulated by sunlight in the facial epidermis of most women during their daily lives, and may be an etiological factor in photoaging, e.g., by promoting wrinkle formation.

Unsaturated fatty acids induce calcium influx into keratinocytes and cause abnormal differentiation of epidermis.

Abnormal follicular keratinization is involved in comedogenesis in acne vulgaris. We recently demonstrated that calcium influx into epidermal keratinocytes is associated with impaired skin barrier function and epidermal proliferation. Based on these results, we hypothesized that sebum components affect calcium dynamics in the keratinocyte and consequently induce abnormal keratinization. To test this idea, we first observed the effects of topical application of sebum components, triglycerides (triolein), saturated fatty acids (palmitic acid and stearic acid), and unsaturated fatty acids (oleic acid and palmitoleic acid) on hairless mouse skin. Neither triglyceride nor saturated fatty acids affected the skin surface morphology or epidermal proliferation. On the other hand, application of unsaturated fatty acids, oleic acid, and palmitoleic acid induced scaly skin, abnormal keratinization, and epidermal hyperplasia. Application of triglycerides and saturated fatty acids on cultured human keratinocytes did not affect the intracellular calcium concentration ([Ca(2+)](i)), whereas unsaturated fatty acids increased the [Ca(2+)](i) of the keratinocytes. Moreover, application of oleic acid on hairless mouse skin induced an abnormal calcium distribution in the epidermis. These results suggest that unsaturated fatty acids in sebum alter the calcium dynamics in epidermal keratinocytes and induce abnormal follicular keratinization.

gamma-Aminobutyric acid (A) receptor agonists accelerate cutaneous barrier recovery and prevent epidermal hyperplasia induced by barrier disruption.

gamma-Aminobutyric acid, is an amino acid transmitter, which mediates rapid inhibition in the central nervous system. gamma-Aminobutyric acid (A) receptor is a ligand-gated chloride ion channel playing an important part in polarizing the cell membrane and reducing neuronal excitability in the neuron. In this study, we demonstrated the effects of gamma-aminobutyric acid (A) receptor agonists on the cutaneous barrier repair process after the barrier disruption of hairless mice. Topical application of gamma-aminobutyric acid and gamma-aminobutyric acid (A) receptor-specific agonists, musimol and isoguvacine, after barrier disruption accelerated the barrier recovery. The gamma-aminobutyric acid (B)-specific agonist, baclofen, did not affect the barrier recovery rate. The effect of gamma-aminobutyric acid on the barrier recovery was blocked by the gamma-aminobutyric acid (A)-receptor antagonist, bicuculline methobromide, but gamma-aminobutyric acid (B) receptor antagonist, saclofen, did not affect the effect of gamma-aminobutyric acid. Topical application of gamma-aminobutyric acid also prevented epidermal hyperplasia, which was induced by the barrier insults under low environmental humidity and bicuculline methobromide blocked the effect of gamma-aminobutyric acid on the epidermal hyperplasia. Immunoreactivity against gamma-aminobutyric acid (A) polyclonal antibody was observed in hairless mouse epidermis. The fluorescent probe of gamma-aminobutyric acid (A) receptor, TXR-musimol showed the localization of gamma-aminobutyric acid (A) receptor in the epidermis of the hairless mice. Elevation of intracellular chloride ion was induced by gamma-aminobutyric acid in cultured human keratinocytes and it was blocked by bicuculline methobromide. These results suggest that the gamma-aminobutyric acid (A)-like receptor is associated with skin barrier homeostasis and regulation of the receptor clinically effective for barrier dysfunctional or epidermal hyperproliferative diseases.

Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse.

A number of studies indicate that matrix metalloproteinase might be involved in photoaging, but little is known about their direct contribution to ultraviolet-induced histologic and morphologic changes in the skin in vivo. This study reports the relationship between changes of matrix metalloproteinase activities and ultraviolet B-induced skin changes in hairless mouse. The role of matrix metalloproteinase in the skin changes was studied by topical application of a specific matrix metalloproteinase inhibitor. The backs of mice were exposed to ultraviolet B three times a week for 10 wk. Histologic studies showed that the basement membrane structure was damaged, with epidermal hyperplasia, in the first 2 wk of ultraviolet B irradiation, followed by the appearance of wrinkles, which gradually extended in the latter half of the ultraviolet B irradiation period. We observed enhancement of type IV collagen degradation activity, but not collagenase or matrix metalloproteinase-3 activity, in extracts of ultraviolet B-irradiated, wrinkle-bearing skin. Gelatin zymographic analysis revealed that gelatinases, matrix metalloproteinase-9 and matrix metalloproteinase-2, were significantly increased in the extract. In situ zymographic study clarified that the activity was specifically localized in whole epidermis of ultraviolet B-irradiated, wrinkled skin in comparison with normal skin. The activity was induced around the basal layer of the epidermis by a single ultraviolet exposure of at least one minimal erythema dose. Furthermore, topical application of a specific matrix metalloproteinase inhibitor, CGS27023A, inhibited ultraviolet B-induced gelatinase activity in the epidermis, and its repeated application prevented ultraviolet B-induced damage to the basement membrane, as well as epidermal hyperplasia and dermal collagen degradation. Ultraviolet B-induced wrinkles were also prevented by administration of the inhibitor. These results, taken together, suggest that ultraviolet B-induced enhancement of gelatinase activity in the skin contributes to wrinkle formation through the destruction of basement membrane structure and dermal collagen in chronically ultraviolet B-exposed hairless mouse, and thus topical application of matrix metalloproteinase inhibitors may be an effective way to prevent ultraviolet B-induced wrinkle formation.

Matrix metalloproteinase-2 inhibition and Zn2+-chelating activities of pyoverdine-type siderophores.

Pyoverdine-type siderophores from fluorescent Pseudomonas species were purified by Zn2+-chelate chromatography, and their matrix metalloproteinase-2 (MMP-2) inhibition and metal-ion-chelating activities were studied. Structurally different pyoverdines showed different MMP-2 inhibition activities, and the inhibition activity was correlated with Zn2+-chelating activity. The IC50 value of a pyoverdine ((P113A1)-2, MW 1187) for MMP-2 was 0.27 microg/ml (0.23 microM).

Formation of Isomeric Tetrathiotungstate Clusters [{Cp*Ru(CO)}2 (WS4 ){W(CO)4 }] by the Reaction of [Cp*2 Ru2 S4 ] with [W(CO)3 (MeCN)3 ].

Thermal and photochemical interconversion occurs between the isomeric pair of tetrathiotungstate [WS4 ]2- clusters 1 and 2, which were formed by thermolysis of [Cp*2 Ru2 S4 ] and [W(CO)3 (MeCN)3 ] [Eq. (1)] and then structurally characterized. During synthesis, a dramatic redistribution of ligands between the Ru and W atoms takes place without the loss of any CO and S ligands.

Safety assurance of cosmetics in Japan: current situation and future prospects.

The Japanese Pharmaceutical Affairs Law distinguishes cosmetics from quasi-drugs, and specifies that they must have a mild effect on the human body and must be safe to use over the long term. Therefore, the safety of cosmetics needs to be thoroughly evaluated and confirmed, taking into account the type of cosmetic, application method, conditions of use and so on. Post-marketing surveys of customers' complaints and case reports of adverse effects are important to monitor and confirm the safety of products. Although manufacturing and marketing of cosmetics are becoming more globalized, the regulations relevant to cosmetics safety still vary from country to country. Thus, compliance with different regulations in various markets is a major issue for producers. In particular, further development of alternatives to animal testing remains an urgent global issue.

Large-scale production of saikosaponins through root culturing of Bupleurum falcatum L. using modified airlift reactors.

Modification of internal configuration of a bubble column, airlift and stirred tank reactor (10-200 L) was made for root cultures of Bupleurum falcatum L. Agitation with an impeller covered with partition mesh was ineffective for a 10-L modified reactor, because it caused intensive foaming and subsequent overflow of the culture medium even at a low rotation speed of 50 rpm and a low aeration rate of 0.1 vvm (volume per volume of medium). In contrast, efficient aeration through a ceramic sparger placed at the bottom of a 20-L bubble column reactor yielded approximately 25 g/L of dry roots and 500 mg/L of saikosaponin-a and saikosaponin-d over 42 days. On a 200-L scale, however, the roots became flocculated under the upper perforated plate initially positioned near the middle of the reactor, forming a firm disk of roots and a large empty space between the disk and the medium. Thus, the roots had poor contact with the medium, which severely suppressed their growth. To avoid this flocculation, a bottom perforated plate and draft tube were installed as a partitioning device separating the culturing area (outside the draft tube) from the aeration area (inside the draft tube). The draft tube was made of a stainless steel mesh rather than a solid material, and the tube greatly increased the root yield in the 20-L reactor. This configuration was successfully applied at the 200-L scale, yielding 500-600 mg/L of saikosaponin-a and saikosaponin-d over 56 days.

Coenzyme Q10 protects against oxidative stress-induced cell death and enhances the synthesis of basement membrane components in dermal and epidermal cells.

Coenzyme Q10 (CoQ10), which has both energizing and anti-oxidative effects, is also reported to have antiaging action, e.g., reducing the area of facial wrinkles. However, the mechanism of its anti-aging activity is not fully established. Here, we examined the effect of CoQ10 on human dermal and epidermal cells. CoQ10 promoted proliferation of fibroblasts but not keratinocytes. It also accelerated production of basement membrane components, i.e., laminin 332 and type IV and VII collagens, in keratinocytes and fibroblasts, respectively; however, it had no effect on type I collagen production in fibroblasts. CoQ10 also showed protective effects against cell death induced by several reactive oxygen species in keratinocytes, but only when its cellular absorption was enhanced by pretreatment of the cells with highly CoQ10-loaded serum. These results suggest that protection of epidermis against oxidative stress and enhancement of production of epidermal basement membrane components may be involved in the antiaging properties of CoQ10 in skin.