Fast liquid chromatography-mass spectrometry glutathione measurement in whole blood: micromolar GSSG is a sample preparation artifact.

We describe a new, fast (6 min) and reliable method to measure reduced or oxidized glutathione (GSH) or (GSSG) in whole blood. The method is based on a LC/MS measurement in positive electrospray ionization mode after a chromatographic separation on a specific column which does not need any counter-ion in the mobile phase, improving the sensitivity of detection. A 50 microl sample of whole blood is sufficient for analysis.

Diaminonaphtalene, a new highly specific reagent for HPLC-UV measurement of total and free malondialdehyde in human plasma or serum.

We describe a new method to measure free and total malondialdehyde (MDA) in human plasma or serum, which is based on the derivatization of MDA with diaminonaphtalene (DAN) in an acidic medium at 37 degrees C. Derivatization is complete after 180 min at room temperature. By HPLC separation on a C18 column and diode array detection, the diazepinium thus formed exhibits a highly specific UV spectrum with a sharp maximum at 311 nm, which clearly distinguishes MDA from other short-chain aldehydes.

Creatine kinase isoenzymes specificities: histidine 65 in human CK-BB, a role in protein stability, not in catalysis.

Creatine kinases (CK) play a prominent role in cell energy distribution through an energy shuttle between mitochondria and other organelles. Human brain CK was cloned and overexpressed in COS-7 cells. We then deleted His-65 and/or Pro-66 situated near the center of a flexible loop as shown by X-ray crystallography on mitochondrial and cytosolic CK.

Pigmented human skin equivalent--as a model of the mechanisms of control of cell-cell and cell-matrix interactions.

The melanin pigment system in human skin is extraordinarly well developed and assures the photoprotection of the skin against harmful solar radiation. Specific cell-cell interactions between one melanocytes and keratinocytes play a fundamental role in the regulation of melanogenesis and melanin pigementation, the two key elements of this system, giving rise to the concept of a structural, functional collaborative 'epidermal melanin unit,' Early experiments strongly suggested that melanocyte growth and differentiation are regulated by paracrine factors from keratinocytes and other skin cells. In addition, co-culture studies with keratinocytes has shown that the extracellular matrix acts as a local environmental signal for dendrite formation and melanogenesis.

Identification of the creatine binding domain of creatine kinase by photoaffinity labeling.

A new photoaffinity probe with a benzophenone group, N-dibenzylphospho-N'-(4-benzoyl)-benzylguanidine (BzPG), has been synthesized on the basis on our previously described creatine kinase bisubstrate analog. BzPG is also a bisubstrate type analog whose photoinsertion is inhibited by the natural substrates of creatine kinase. When rabbit CK-MM is irradiated in the presence of BzPG then cleaved by CNBr, one labeled peptide can be purified by reverse phase HPLC and sequenced.

Regulatory effects of heat on normal human melanocyte growth and melanogenesis: comparative study with UVB.

Although energy-rich ultraviolet B (UVB) is considered to be primarily responsible for most of the effects associated with solar radiation, small energy recorded as heat appears to contribute to the biologic effects of solar radiation on the skin. We compared the effects of heat and UVB on normal human melanocyte functions. In monolayer culture the following was found.

Pigmented human skin equivalent: new method of reconstitution by grafting an epithelial sheet onto a non-contractile dermal equivalent.

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components.

Measurements of the protective effect of topically applied sunscreens using in vitro three-dimensional dermal and skin equivalents.

For preventing or minimizing acute and chronic skin damage caused by UV radiation, the use of sunscreens is probably the most important measure. To screen the protective efficacy of new sunscreen molecules or formulations against UV rays, we evaluated as in vitro testing methods the use of two three-dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen-glycosaminoglycans-chitosan matrix populated by normal human fibroblasts.

N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)propyl-guanidine is a bisubstrate-analog for creatine kinase.

We describe a new compound, N-dibenzylphospho-N'-3-(2,6-dichlorophenyl)-propylguanidine (DPPG), and our study of its interaction with cytosolic CK. To our knowledge, it is the most potent inhibitor known for CK: the Ki value versus ADP was 330 nM and 110 nM for CK-MM and BB respectively. In view of the inhibition pattern, Ki(app) dependencies on the second substrate, and very low Ki values, we conclude that DPPG binds to the active site as a bisubstrate-type analog.

Simultaneous measurement of seven carotenoids, retinol and alpha-tocopherol in serum by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (HPLC) procedure for the quantitative measurement in serum of seven carotenoids (lutein, zeaxanthin, canthaxanthin, beta-cryptoxanthin, lycopenes, alpha-carotene and beta-carotene), retinol, alpha-tocopherol and two internal standards (tocol and echinenone) has been developed. The geometric isomers, lutein and zeaxanthin, were completely separated as well as at least nine unidentified carotenoids. All compounds were resolved on an Adsorbosphere HS C18 (3 microm) column (250x4.

Dichloroaromatic phosphoguanidines are potent inhibitors but very poor substrates for cytosolic creatine kinase.

New phosphorylated guanidines have been synthesized and examined as potential inhibitors for creatine kinase. These compounds show a significant increase of inhibitory activity in comparison with the corresponding guanidines. Unlike the guanidines, they are competitive inhibitors because of the phosphoryl group.

Use of dermal equivalent and skin equivalent models for identifying phototoxic compounds in vitro.

Phototoxicity inducing in vivo photoirritation, a reversible inflammatory reaction of the skin after chemical contact and UVA radiation exposure, is increasingly observed as a side effect associated with the use of both cosmetics and systemic drugs. In order to systematically screen for the phototoxic potential of new compounds, we propose two three-dimensional models suitable for in vitro testing: a dermal equivalent (DE) and a skin equivalent (SE) model. The DE model includes a collagen-glycosaminoglycans-chitosan porous matrix populated by normal human fibroblasts.

Deposition of collagen fibril bundles by long-term culture of fibroblasts in a collagen sponge.

Human fibroblasts cultured for 10 days in a collagen sponge migrated through the pores of the sponge and expressed a moderate mitotic activity, which stabilized after 10 days, and a high collagen and protein synthesis. Between 10 and 27 days, the newly synthesized collagen filled the pores of the sponge. This matrix accumulation induced a delayed decrease of collagen and protein synthesis.

Synthesis and differential properties of creatine analogues as inhibitors for human creatine kinase isoenzymes.

Fourteen new creatine analogues, all with a guanidine function and either a polar or an apolar group instead of the creatine carboxylic function, were tested as potential inhibitors for human creatine kinase by kinetic analysis of their effects on the reaction rate. Only compounds bearing an apolar aromatic moiety, which was spaced from the guanidine function by at least two bonds, proved to have a significant inhibitory activity and showed a mixed-type inhibition similar to that of creatine. Among these compounds 2,6-dichlorobenzylguanidine (Ki = 5.

Effects of calphostin C, specific PKC inhibitor on TPA-induced normal human melanocyte growth, morphology and adhesion.

Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate-stimulating agents or 12-0-tetradecanoylphorbol 13-acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)-activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC-mediated pathway in the TPA-dependent melanocyte functions-i.

Mesenchymal-epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal-epidermal junction formation in a skin equivalent model.

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal-epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal-epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact.

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.

Keratinocyte extracellular matrix-mediated regulation of normal human melanocyte functions.

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system.

A dermal substrate made of collagen--GAG--chitosan for deep burn coverage: first clinical uses.

In cases of severe burns, it seems necessary to excise burnt tissues as soon as possible and to cover the excised area immediately with a skin substitute, when few autografts are available. We report here the first clinical uses of a dermal substrate made of collagen--GAG--chitosan grafted immediately after early excision, then epidermalized either with autologous meshed autograft or with autologous cultured epidermis. The dermal substrate replaces the excised dermis by adhering to the underlying tissue, promoting fibrovascular ingrowth.

Optimization of thickness, pore size and mechanical properties of a biomaterial designed for deep burn coverage.

A collagen and chondroitins 4-, 6-sulphate biomaterial designed for the coverage of severe burns was optimized in terms of mechanical strength by addition of 20% (wt/vol) of chitosan to the starting material. Chitosan should create ionic bonds with collagen and thus increase the tensile strength and Young's modulus of the sponge. On the other hand, sterilization by h-irradiation of the biomaterial induced a decrease in its mechanical properties that could be avoided by sterilization using beta-irradiation.

Modulation of normal human melanocyte dendricity by growth-promoting agents.

Dendrite formation and extension, which comprise a characteristic morphology of human normal melanocytes in the skin, represent one of the functional activities of melanocytes, the ability to transfer melanosomes into neighboring keratinocytes. However, the morphology of the melanocyte in vitro is usually quite different from that observed in vivo. it is probably due to the hyperproliferative condition of the melanocytes in culture.

Collagen synthesis by fibroblasts cultured within a collagen sponge.

We prepared a collagen sponge made of type I and III bovine collagen, glycosaminoglycans (GAG) and chitosan. Fibroblasts grown within the collagen sponge express a sixfold increase of their collagen synthesis, compared with fibroblasts embedded in a collagen gel. Moreover, collagen synthesis is twice as high in the collagen sponge than in a monolayer culture.

Reconstruction of epidermis on a chitosan cross-linked collagen-GAG lattice: effect of fibroblasts.

Reconstruction of epidermis on a fibroblast containing chitosan cross-linked collagen-GAG lattice at the air-liquid interface gives rise to a multilayered stratified epithelium, covered with a compact stratum corneum. Immunohistological studies reveal that the markers of epidermal differentiation are essentially distributed as in normal human skin and that the major proteins of the dermal-epidermal junction are present. Reconstruction of epidermis under identical culture conditions on a dermal equivalent that does not contain fibroblasts gives rise to an epithelium consisting of disorganized cell layers where the markers of differentiation are either displaced or not at all expressed, as in the case with filaggrin.

Cytotoxicity evaluation of antiseptics and antibiotics on cultured human fibroblasts and keratinocytes.

Infection is the greatest problem in burn patients and topical antimicrobial agents must be chosen with great care, especially when cultured skin is grafted. We examined the cytotoxic effect of six antiseptics and six antibiotics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane (chlorhexidine), Biseptine (chlorhexidine+benzalkonium chloride+benzylic alcohol), Benzalkonium Chloride, Yellow Betadine (polyvidone-iodine+nonoxinol), Betadine Scrub (polyvidone-iodine+quaternary ammonium) and Green Betadine (polyvidone-iodine) and viability was determined using the MTT test.