Biphasic effect of UVA radiation on STAT1 activity and tyrosine phosphorylation in cultured human keratinocytes.

The effect of ultraviolet A (UVA) radiation on the DNA-binding activity of the transcription factor STAT1 was studied by electromobility shift assay in the human keratinocyte cell line NCTC 2544. The STAT1-binding activity exhibited a biphasic pattern as a function of UVA doses. For UVA doses lower than 0.6 J/cm(2), a dose-dependent increase in STAT1 activity was observed. In a second phase, with higher UVA doses (1.5 to 9 J/cm(2)), the activity decreased and reached control value at 6 J/cm2. The enhancement of STAT1 activity was transient, peaked at 1 h after UV irradiation, and regularly decreased to control value 24 h after UV. Genistein, a tyrosine kinase inhibitor, H7, a serine/threonine kinase inhibitor, and PD 98059, a MEK inhibitor, prevented the UVA-induced enhancement of STAT1-binding activity, suggesting the involvement of Tyr, Ser/Thr kinases, and MEK in the observed effect. Immunoblot analysis directly demonstrated that the amount of Tyr-phosphorylated STAT1 was parallel to its DNA-binding activity. Immunoblot analysis also demonstrated the nuclear transport of STAT1 after UVA irradiation at low doses. At high doses, a decrease in the STAT1 level was observed both in the cytoplasmic and the nuclear compartments, suggesting that the inactivation was due to a degradation process. UVA irradiation initiated a dose-dependent increase in lipid peroxidation products and reactive oxygen species. Furthermore, the involvement of the oxidative stress in the UVA-induced effect on STAT1 activity is suggested by the protective action of the antioxidants alpha-tocopherol and N-acetylcysteine on both the activation phase (UVA doses lower than 1.5 J/cm(2)) and the inhibitory phase. By contrast, the pro-oxidant drug buthionine sulfoximine enhanced the effect of UVA on STAT1-binding activity. Since STATs are known as transducers of cytokine action, the enhancement of STAT1 activity by low doses of UVA might be related to the proinflammatory effect of solar radiations at the skin level.

Oxidized LDL activates STAT1 and STAT3 transcription factors: possible involvement of reactive oxygen species.

The effect of cupric ion-oxidized low density lipoprotein (Cu-LDL) or endothelial cell-oxidized LDL (E-LDL) on STAT1 and STAT3 (signal transducers and activators of transcription) DNA binding activity was investigated by electrophoretic mobility shift assay in human endothelial cells. Both oxidized LDL enhanced STAT1 and STAT3 binding to their respective consensus binding sites. Furthermore, the activation of STATs was proportional to the oxidation degree of LDL in that the highly oxidized Cu-LDL exhibited a more marked effect than E-LDL. Oxidized LDL induced an intracellular oxidative stress, as shown by the increase in the intracellular level of lipid peroxidation products (thiobarbituric acid-reactive substances) and in the level of reactive oxygen species, measured by the fluorescence of dichlorofluorescein diacetate. The binding activity of STAT1 and STAT3 paralleled these two parameters, which suggests that it is dependent upon the redox state of the cell. The activation of STATs by oxidized LDL was almost completely inhibited by the lipophilic antioxidant vitamin E, and partially antagonized by the hydrophilic thiol-containing compound N-acetylcysteine, suggesting that the oxidative stress induced by oxidized LDL is involved in the observed phenomenon. Furthermore, the lipid extract of Cu-LDL also activated STAT1 and STAT3. Since the STAT pathway plays a key role in cytokine and growth factor signal transduction, the activation of STATs by oxidized LDL might be related to their proinflammatory and fibroproliferative effect in the atherosclerotic plaque.

Lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells.

The effect of lipopolysaccharide (LPS, endotoxin) on low density lipoprotein (LDL) oxidative modification by copper ions, endothelial and smooth muscle cells was studied by determination of the level of lipid peroxidation products (thiobarbituric acid reactive substances or TBARS), the diene level and the electrophoretic mobility of the LDL particle. LPS 25-75 microg/ml induced a dose-dependent increase in LDL oxidation by copper ions, endothelial and smooth muscle cells. At 75 microg LPS/ml, the TBARS content was 1.9, 1.6, and 1.8-fold increased, respectively. The LDL degradation by J774 macrophage-like cells was concomitantly stimulated. Preincubation of the LDL particle with LPS induced a marked increase in the subsequent LDL oxidative modification either by copper ions or by endothelial and smooth muscle cells. In addition, pretreatment of endothelial and smooth muscle cells with LPS also induced an enhancement of LDL oxidative modification performed in the absence of LPS. This effect was accompanied by a parallel increase in superoxide anion release by the cells. These results point at one of the mechanisms involved in the described association between bacterial infection and acute myocardial infarction as well as coronary heart disease.

Quantification of HTLV type I and HIV type I DNA load in coinfected patients: HIV type 1 infection does not alter HTLV type I proviral amount in the peripheral blood compartment.

Several reports suggest that HTLV-I/HIV coinfection may be associated with an increased risk of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In HTLV-I-monoinfected patients, the occurrence of HAM/TSP is associated with high peripheral blood HTLV-I proviral load. Using a real-time quantitative PCR assay, we assessed the proviral DNA load in peripheral blood mononuclear cells (PBMCs) from 15 asymptomatic HTLV-I-monoinfected patients, 15 HTLV-I-monoinfected patients with HAM/TSP, and 25 HTLV-I/HIV-1 coinfected patients, including 4 with HAM/TSP. We also measured HIV-1 proviral DNA load in PBMCs from the coinfected patients. The median HTLV-I proviral loads were 6,800 and 4,100 copies per 10(6) PBMCs in the asymptomatic monoinfected and coinfected groups, and 58,800 and 43,300 copies per 10(6) PBMCs in the monoinfected and coinfected patients with HAM/TSP, respectively. The difference between HTLV-I proviral loads in HAM/TSP and asymptomatic monoinfected patients was statistically significant (p < 0.0001), but there was no difference between the HTLV-I-monoinfected and HTLV-I/HIV-1-coinfected groups. There was no correlation between HTLV-I and HIV-1 proviral load. HTLV-I proviral load did not correlate with the CD4+ T lymphocyte count. Among patients with no HTLV-I disease, the median copy number of HTLV-I per 10(6) circulating CD4+ T cells was 114,000 in the coinfected group and 16,700 in the monoinfected group, but the difference was not significant (p = 0.089). These data do not confirm the hypothesis in which HIV-1 coinfection would increase HTLV-I proviral burden in the PBMCs. However, depletion of the CD4+ T cell subset, the main target of HTLV-I, could be counterbalanced by an up-regulation of HTLV-I replication or by greater resistance of HTLV-I-infected cells to HIV-1-induced destruction.

Mapping the subcellular distribution of biomolecules at the ultrastructural level by ion microscopy.

Analytical ion microscopy, a method proposed and developed in 1960 by Casting and Slodzian at the Orsay University (France), makes it possible to obtain easily and rapidly analytical images representing the distribution in a tissue section of elements or isotopes (beginning from the three isotopes of hydrogen until to transuranic elements), even when these elements or isotopes are at a trace concentration of 1 ppm or less. This method has been applied to study the subcellular distribution of different varieties of biomolecules. The subcellular location of these molecules can be easily determined when the molecules contain in their structures a specific atom such as fluorine, iodine, bromine or platinum, what is the case of many pharmaceutical drugs. In this situation, the distribution of these specific atoms can be considered as representative of the distribution of the corresponding molecule. In other cases, the molecules must be labelled with an isotope which may be either radioactive or stable. Recent developments in ion microscopy allow the obtention of their chemical images at ultra structural level. In this paper we present the results obtained with the prototype of a new Scanning Ion Microscope used for the study of the intracellular distribution of different varieties of molecules: glucocorticoids, estrogens, pharmaceutical drugs and pyrimidine analogues.

Oxidized LDL induces an oxidative stress and activates the tumor suppressor p53 in MRC5 human fibroblasts.

It is now well established that oxidized LDL (OxLDL) is involved in the progression of the atheromatous plaque via several mechanisms, including its cytotoxicity toward the arterial wall. Our study demonstrates that a 4-h incubation of cultured human fibroblasts with 25-75 microg/ml OxLDL induced a dose-dependent increase in the intracellular levels of reactive oxygen species (ROS) and lipid peroxidation end products (TBARS). This effect was markedly prevented by the antioxidant vitamin E. The lipid extract of OxLDL partially reproduced the action of the LDL particle itself. Concomitantly, OxLDL enhanced the DNA binding activity of p53 measured by electrophoretic mobility shift assay, and the intracellular protein level of p53 determined by immunoblot analysis. Cycloheximide prevented the OxLDL-induced augmentation in both p53 binding activity and intracellular level. Again, the lipid extract of OxLDL reproduced the effect of OxLDL on p53 binding activity, whereas vitamin E prevented it. These results indicate that OxLDL initiates an intracellular oxidative stress by means of its lipid peroxidation products, leading to the activation of the tumour suppressor p53 by enhancement of p53 protein synthesis. This effect might be related to the cytotoxic effect of OxLDL since the activation of p53 is known to lead to cell cycle arrest, necrosis or apoptosis.

Scanning ion microscopy mapping of basement membrane elements and arterioles in the kidney after selenium-silver interaction.

The effects of selenium and silver salts was studied by scanning ionic microscopy during experimental argyria mapping of the different basement membrane elements. The ionic microscope (IMS 4F) was equipped with a high resolution spectrometer giving high spatial resolution on the image obtained. After long-term treatment with silver salt alone, silver and sulphur deposits were observed in the membranes. After administration of selenium and silver salt, it was possible to map nitrogen, sulphur, selenium and silver to the glomerular basement membrane as well as to the wall of the kidney arterioles. In the latter, sulphur, selenium and silver were localized only in the elastic laminae of the walls. This process of precipitation of silver deposits in the membrane can be interpreted as process of selenium "detoxification" of the organism.

Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation.

Antisense transgenesis of tobacco (Nicotiana tabacum) with a partial flax (Linum usitatissimum L.) pectin methylesterase (Lupme3) cDNA sequence yielded plants with altered pollen content. Moreover, the characteristically sculptured cell wall surrounding the pollen grains was modified in transgenic tobacco plants: the wavy ornamentation was dramatically reduced, suggesting the involvement of the demethylation of pectin in the pollen cell wall-specific structure. Germination of pollen was decreased and the pollen tube surface aspect was also different in transgenic plants.

Polyunsaturated fatty acid enrichment enhances endothelial cell-induced low-density-lipoprotein peroxidation.

Oxidative modification of low-density lipoprotein (LDL) is an important feature in the initiation and progression of atherosclerosis. LDL modification by endothelial cells was studied after supplementation of the cells with oleic acid and polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series. In terms of the lipid peroxidation product [thiobarbituric acid reactive substances (TBARS)] content and diene level of the LDL particle, oleic acid had no significant effect, and linoleic acid was poorly effective. Gamma linolenic acid (C18:3,n-6) and arachidonic acid (C20:4,n-6) increased by about 1.6-1.9-fold the cell-mediated LDL modification. PUFA from the n-3 series, alpha linolenic acid (C18:3,n-3), eicosapentaenoic acid (C20:5,n-3) and docosahexaenoic acid (C22:6,n-3), induced a less marked effect (1. 3-1.6-fold increase). The relative electrophoretic mobility of the LDL particle and its degradation by macrophages were enhanced in parallel. Concomitantly, PUFA stimulated superoxide anion secretion by endothelial cells. The intracellular TBARS content was also increased by PUFA. Comparison of PUFA from the two series indicates a good correlation between LDL oxidative modification, superoxide anion secretion and intracellular lipid peroxidation. The lipophilic antioxidant vitamin E decreased the basal as well as the PUFA-stimulated LDL peroxidation. These results indicate that PUFAs with a high degree of unsaturation of the n-6 and n-3 series could accelerate cell-mediated LDL peroxidation and thus aggravate the atherosclerotic process.