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51.
This study was implemented to test the Episkin model of reconstructed epidermis in the evaluation of the efficacy of cosmetic or dermopharmaceutical products on cutaneous energy metabolism. The energy metabolism is evaluated by measuring the concentration of intracellular ATP by a method using an ultrasensitive bioluminescent reaction. The work presented compares results obtained in reconstructed epithelium and monolayer primary cultures of human keratinocytes.After application of a hydrosoluble product, the increase in intracellular ATP is identical in a monolayer culture of keratinocytes (+239±18% versus control) and in Episkin (+248±21% versus control). An emulsion was also tested on the two models. It is only possible to test the emulsion at a dilution of under 0.05% on a keratinocyte culture, and this means that the real efficacy of the product is underestimated (+145±18% versus control). The three-dimensional model enables the application of the undiluted emulsion, and the results show an increase in intracellular ATP of +420±80% versus control: products in final formulation can be tested in normal conditions of use.Abbreviations BPE
bovine pituitary extract
- DMEM
Dulbecco's modified Eagle's medium
- EDTA
ethylene diamine tetraacetic acid
- EGF
epidermal growth factor
- K-SFM
keratinocytes serum free medium
- MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
- O/W
oil in water
- PBS
phosphate-buffered saline 相似文献
52.
Olivier Bornet Gérard Lancelot Luc Chanteloup Nguyen T. Thuong Jean-Marie Beau 《Journal of biomolecular NMR》1994,4(4):575-580
Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively 13C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the 1H1–13C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum. 相似文献
53.
Alain Gerbi Marcel Debray Jean-Michel Maixent† Claude Chanez Jean-Marie Bourre 《Journal of neurochemistry》1993,60(1):246-252
Abstract: The Na+ sensitivity of whole brain membrane Na+ ,K+ -ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na+ , we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10−5 and 10−7 M ), we were able to discriminate Na+ sensitivity of Na+ , K+ -ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na+ sensitivity [ K a of 3.88 ± 0.25 m M Na+ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na+ sensitivities, a high ( K a of 4.98 ± 0.2 m M Na+ and n of 1.34 ± 0.10) and a low ( K a of 28 ± 0.5 m M Na+ and an n of 1.92 ± 0.18) Na+ sensitivity activated above a thresh old (22 ± 0.3 m M Na+ ); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na+ sensitivities (apparent K a values of 3.5 and 20 m M Na+ ). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes. 相似文献
54.
55.
The MO15 gene encodes the catalytic subunit of a protein kinase that activates cdc2 and other cyclin-dependent kinases (CDKs) through phosphorylation of Thr161 and its homologues. 总被引:43,自引:15,他引:28 下载免费PDF全文
D Fesquet J C Labb J Derancourt J P Capony S Galas F Girard T Lorca J Shuttleworth M Dore J C Cavadore 《The EMBO journal》1993,12(8):3111-3121
Phosphorylation of Thr161, a residue conserved in all members of the cdc2 family, has been reported to be absolutely required for the catalytic activity of cdc2, the major regulator of eukaryotic cell cycle. In the present work, we have purified from starfish oocytes a kinase that specifically activates cdc2 in a cyclin-dependent manner through phosphorylation of its Thr161 residue. Our most highly purified preparation contained only two major proteins of apparent M(r) 37 and 40 kDa (p37 and p40), which could not be separated from each other without loss of activity. The purified kinase was found to phosphorylate not only cdc2, but also cdk2 and a divergent cdc2-like protein from Caenorhabditis, in chimeric complexes including both mitotic and G1/S cyclins. Extensive microsequencing of p40 did not reveal any convincing homology with any known protein. In contrast, p37 is the starfish homologue of the M015 gene product, a kinase previously cloned by homology probing from a Xenopus cDNA library. As expected, immunodepletion of the MO15 protein depleted Xenopus egg extracts of CAK (cdk-activating kinase) activity, which was recovered in immunoprecipitates. Taken together, the above results demonstrate that MO15 is a gene conserved throughout evolution (at least from echinoderms to vertebrates) that encodes the catalytic subunit of a protein kinase that activates cdc2-cdks complexes through phosphorylation of Thr161 (or its homologues). 相似文献
56.
Holger Stephan Stefan Freund Werner Beck Günther Jung Jean-Marie Meyer Günther Winkelmann 《Biometals》1993,6(2):93-100
Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn1(N
-OH, N
-acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn4(N
-OH, N
-formyl)-1,4-diaminobutane. The N
-acyl groups of Orn1(N
-OH, N
-acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques. 相似文献
57.
A portion of spinal cord taken from a pig infected with the Konratice strain of Teschen Disease virus was found to be infectious for swine after an eleven year period of storage at dry ice temperature. The virus was recovered in tissue culture from the brains of two experimentally infected pigs, titrated, and a serum-virus neutralization test performed. 相似文献
58.
Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells 总被引:1,自引:0,他引:1
Olivier Rabin Michèle Piciotti Katy Drieu Jean-Marie Bourre Françoise Roux 《In vitro cellular & developmental biology. Animal》1996,32(4):221-224
Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE4 cells). A sublethal anoxic period of 12 h was assessed for RBE4 cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase,
with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity
modulated by the presence or absence of oxygen returned to control value.
Damage and recovery of RBE4 immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides
a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level. 相似文献
59.
The serum response factor nuclear localization signal: general implications for cyclic AMP-dependent protein kinase activity in control of nuclear translocation. 总被引:5,自引:1,他引:4 下载免费PDF全文
C Gauthier-Rouvire M Vandromme N Lautredou Q Q Cai F Girard A Fernandez N Lamb 《Molecular and cellular biology》1995,15(1):433-444
We have identified a basic sequence in the N-terminal region of the 67-kDa serum response factor (p67SRF or SRF) responsible for its nuclear localization. A peptide containing this nuclear localization signal (NLS) translocates rabbit immunoglobulin G (IgG) into the nucleus as efficiently as a peptide encoding the simian virus 40 NLS. This effect is abolished by substituting any two of the four basic residues in this NLS. Overexpression of a modified form of SRF in which these basic residues have been mutated confirms the absolute requirement for this sequence, and not the other basic amino acid sequences adjacent to it, in the nuclear localization of SRF. Since this NLS is in close proximity to potential phosphorylation sites for the cAMP-dependent protein kinase (A-kinase), we further investigated if A-kinase plays a role in the nuclear location of SRF. The nuclear transport of SRF proteins requires basal A-kinase activity, since inhibition of A-kinase by using either the specific inhibitory peptide PKIm or type II regulatory subunits (RII) completely prevents the nuclear localization of plasmid-expressed tagged SRF or an SRF-NLS-IgG conjugate. Direct phosphorylation of SRF by A-kinase can be discounted in this effect, since mutation of the putative phosphorylation sites in either the NLS peptide or the encoded full-length SRF protein had no effect on nuclear transport of the mutants. Finally, in support of an implication of A-kinase-dependent phosphorylation in a more general mechanism affecting nuclear import, we show that the nuclear transport of a simian virus 40-NLS-conjugated IgG or purified cyclin A protein is also blocked by inhibition of A-kinase, even though neither contains any potential sites for phosphorylation by A-kinase or can be phosphorylated by A-kinase in vitro. 相似文献
60.
The TEM-1 β-lactamase is a globular protein containing 12 proline residues. The folding mechanism of this enzyme was investigated by kinetic and equilibrium experiments with the help of fluorescence spectroscopy and circular dichroism. The equilibrium denaturation of the protein induced by guanidine hydrochloride occurs in two discrete steps, indicating the existence of a thermodynamically stable intermediate state. Thisstate is 5.2 ± 0.4 kcal/mol less stable than the native conformation and 5.7 ± 0.2 kcal/mol more stable than the fully denaturedprotein. This intermediate state exhibits a high content of native secondary structure elements but is devoid of specific tertiary organization; its relation to the “molten globule” is discussed. Refolding kinetic experimentsrevealed the existence of a transient intermediate conformation between thethermodynamically stable intermediate and the native protein. This transient intermediate appears rapidly during the folding reaction. It exhibits a secondary structure content very similar to that of the native protein and has also recovered a significant amount of tertiary organisation. The final refolding step of the TEM-1 β-lactamase, leading to the native enzyme, is dominated by two major slow kinetic phases which probablyreflect a very complex process kinetically limited by proline cis/transisomerization. © 1995 Wiley-Liss, Inc. 相似文献