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. Direct injection of deproteinized plasma avoids the use of an internal standard. The between-run imprecision is 9.1% (141 +/- 13 nM) for plasma and 6.6% (658 +/- 44 nM) for a commercial control. Typical within-day imprecision is 8% (93 +/- 7.5 nM) for total MDA, 3.2% (16 +/- 0.5 nM) for free MDA in plasma, and 1.6% (630 +/- 10 nM) for a commercial control. The recovery of MDA added to 10 different plasmas is 93-108% (mean = 100%). Plasma levels in healthy women (n = 79, 45-51 years) are 162 +/- 51 and 24 +/- 15 nM for total and free MDA, respectively. In younger men (n = 19, 21-37 years) total and free MDA are, respectively, 138 +/- 28 and 19 +/- 9 nM.

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. The DeltaH65 mutant had nearly the same affinity for its substrates as wild isoenzyme, but its stability was very low. Unlike DeltaH65, DeltaH65P66 had a eightfold decreased affinity for creatine phosphate and was unable to dephosphorylate cyclocreatine phosphate. Our results demonstrate that, despite an overall similar shape of the proteins, this loop accounts for some subtle differences in isoenzyme functions.

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. This sequence of 31 amino acids (Ala30-Val60) contains a region which could be responsible for isoenzyme selectivity and another one just preceding the 11 amino acid peptide (Asp61-Thr70) very recently described as a putative creatine binding site. This second peptide was deduced from the comparison of 18 amino acid sequence alignments. We proposed the creatine binding site to be essentially a peptide from Lys39 to Val71.

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. (i) Heat-treated melanocytes showed an increased dendricity and exhibited a larger cell body compared with nontreated melanocytes. (ii) After multiple treatments with UVB (20 mJ per cm2, 312 nm) or heat (42 degrees C for 1 h) for 3 d, melanocytes had a lower survival than nontreated melanocytes, but they resumed proliferation within 6 d in the same manner as seen in control. (iii) The expression levels of cell cycle regulators, p53 and p21 proteins, were increased after multiple treatments with UVB or heat. (iv) The tyrosinase (dopa-oxidase) activity per cell was increased after the multiple treatments with UVB or heat. (v) The number of dopa-positive melanocytes in coculture with keratinocytes in epithelial sheets was greatly increased by UVB or heat treatments. (vi) Similarly, the increased number of tyrosinase-related protein 1 positive melanocytes was seen in skin equivalents after UVB (100 mJ per cm2) or heat (42 degrees C for 1 h) treatments for 7 d. These results suggest that heat shares significant biologic activities with UVB in melanocyte functions. These results could be considered as one of the protective or adaptive responses of the skin pigmentary system to the environment.

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. Attempts to reconstruct pigmented human skin in vitro have made great progress over the last decade. The behavior of cells in these pigmented human skin equivalents closely resembles that in vivo, and the cells can still respond to appropriate extrinsic regulatory stimuli such as ultraviolet radiation. Keratinocytes and fibroblasts have been shown to be active partners in the regulation of melanocyte distribution, viability and other differentiation functions, presumably by direct contact and the effects of various soluble paracrine factors. By reproducing cell-cell and cell-matrix interactions, these culture systems provide a promising experimental model for investigating regulation of the skin pigmentary system and the role of photoprotection against harmful solar radiation.

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. This bisubstrate analog confirms different mechanisms for CK-MM and BB: in spite of a more than 80% similarity in amino-acid sequences, both isoenzymes are random at pH 8.6 but CK-BB has an ordered mechanism at pH 6.6.

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 (250x4.6 mm I.D.) with a step gradient, 7.1 min after injection, from acetonitrile-methanol (60:40, v/v) containing 0.05% acetic acid to acetonitrile-methanol-dichloromethane (45.6:30.4:24, v/v) containing 0.04% acetic acid, in a total run time of 23 min. Chromatograms at four different wavelengths (292, 325, 450 and 473 nm) and spectra were monitored with a diode array detector. Because of its specificity and sensitivity for a large number of carotenoids, this procedure may be of interest for nutritional and epidemiological studies. Its speed and robustness make it suitable for analyses on large numbers of subjects.

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. N-Phospho-N'-2-(2,6-dichlorophenyl)ethylguanidine is a potent inhibitor (K(i) = 2.0 mM and 1.2 mM respectively for muscle and brain-type creatine kinase). Although these phosphorylated analogs of creatine phosphate have a very poor substrate activity in the reverse reaction, the phosphoryl group is important for binding to the enzyme.

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. The SE model is made by seeding normal human keratinocytes onto the DE, leading to a fully differentiated epidermis. The objectives of this pilot study are: 1) to compare the deleterious effects of UVA radiation on the two models and 2) to evaluate to what extent the in vitro results can predict the in vivo phototoxicity caused by well-known photoirritant compounds, included in the COLIPA validation phototoxicity reference chemical list. Dilutions of thiourea, sulisobenzone, promethazine, chlorpromazine and tetracycline were applied (20 microliters) onto DEs and SEs (n = 6) and incubated for 1 h (or 15 h) at 37 degrees C. Irradiated samples received 3 J/cm2 UVA. The 24 h post-irradiation residual cellular viability was measured using the MTT test on treated and untreated tissues and IL-1 alpha release measurement in collected SE culture media. A concordance in terms of photoirritant/non-photoirritant was obtained between the in vivo data and the in vitro results, suggesting that the DE and the SE models could be integrated, after a complete validation study, into a protocol for in vitro testing of the photoirritant potential of new molecules.

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. The SE is comprised of a fully differentiated epidermis realized by seeding keratinocytes onto the DE. In this study, we demonstrated that the DE and SE models react to the deleterious effects of UVA and UVB. Then, we extended our research to the evaluation of their usefulness for photoprotection trials. Sunscreen agents (Eusolex 8020 and 6300) and commercially available sunscreens (chemical and physical filter formulations) that protect the skin against either UVA or UBV were evaluated. The tested products were applied (n = 6) topically (10 microL) and incubated for 30 min prior to irradiation over a range of UVA (0-50 J/cm2) or UVB (0-5 J/cm2). The photoprotection provided by the tested sunscreen molecules and formulations was evaluated by measurement of residual cellular viability 24 h postirradiation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and assessment of the inflammation response by interleukin-1 alpha release assay. When sunscreens were applied prior to UV exposure, a higher residual cellular viability versus control was obtained, demonstrating the photoprotective effects of the tested products. These in vitro models could be used for screening tests to evaluate the protective effects of sunscreen molecules and formulations, especially for UVA trials because there is a lack of consensus for an in vivo method.

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. ii) Ki67-positive proliferating keratinocytes (18.1 +/- 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm2 per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.

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. Finally, after 27 days of culture, the fibroblasts expressed low biosynthetic activities similar to the ones exhibited in vivo. The newly synthesized matrix was highly differentiated, as shown by the presence of a dense network of quarter-staggered collagen fibrils (42 nm +/- 6 nm in diameter) surrounding the cells. The size and the shape of these fibrils demonstrated that the newly synthesized procollagen was fully processed in collagen by removal of their N- and C-terminal propeptides. Moreover, these fibrils were packed in bundles organized into an interwoven network that mimics the pattern of the papillary dermis. These findings show that fibroblasts cultured for one month in a collagen sponge construct large amounts of a highly differentiated connective tissue.

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 = 5.6 mM and 39.8 mM for muscle-type and brain-type creatine kinases, respectively) and 3-(2,6-dichlorophenyl)propylguanidine (Ki = 15 mM and 4.5 mM) were the more potent inhibitors and showed a significant isoenzyme selectivity between muscle- and brain-type creatine kinases. Our results are in agreement with recent data that suggest the location of a hydrophobic pocket near the guanidine-binding domain of the enzyme. The observed selectivity in isoenzyme inhibition may be useful to study structural differences in catalytic centers.

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.e., proliferation, morphology, and adhesion-using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 +/- 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.

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. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and alpha6 integrin at the dermal-epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase-polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal-epidermal junction by increasing the production and the further arrangement of dermal-epidermal junction components.

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.

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. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs.(ABSTRACT TRUNCATED AT 250 WORDS)

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. The thickness, pore size and morphology of the biomaterial were optimized before freeze-drying by freezing the mixture at -60 degrees C at a weight/volume concentration of 1.25% and a volume of 270 mul/cm2. The biomaterial obtained under these conditions may further the vascularization and cellular colonization of the porous structure by the host cells of the wound bed and therefore may accelerate the regeneration of a new dermis.

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. Then after 15 days it can be epidermalized. The quality of the underlying dermis obtained permitted 100% take after epidermalization with large-meshed autograft, and tended to avoid the usual typical diamond aspect of the meshed skin. After epidermalization with autologous cultured autograft, the quality of the underlying dermis permits a good take. The best aspect is obtained by combining dermal substrate and autologous cultured epidermis. Even if it still does not replace the high quality of a homograft, this dermal substrate is a promising solution for replacement of dermis. It is always available, can be stored and is exempt from micro-organism transmission.