Abnormal production of interleukin (IL)-11 and IL-8 in polycythaemia vera

We studied the production of interleukin (IL)-11 and IL-8, two cytokines known to affect erythropoiesis, in polycythemia vera (PV). In vivo, IL-11 was detected more frequently in serum and bone marrow (BM) plasma of PV patients than in controls (healthy donors and patients with idiopathic erythrocytosis (IE)). In addition, serum IL-11 levels of PV patients were higher than those of controls. IL-8 was elevated in serum of both PV and IE patients (respective median levels: 38.6 and 242pg/ml, vs 4.4pg/ml for healthy donors). BM plasma IL-8 levels of PV patients (508pg/ml) were significantly higher than those of IE patients (120pg/ml). In vitro, bone marrow (BM) stromal cells (BMSC) of PV patients produced significantly more IL-11 (x6.4) and IL-8 (x8.3) than BMSC of healthy donors or IE patients. In conclusion, both IL-11 and IL-8 are overproduced in PV, apparently by BMSC; IL-8 is also overproduced in IE, by cells other than BMSC.     

Hydrogen peroxide generation in caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid

Phenolic compounds have recently attracted special attention due to their beneficial health effects; their intestinal absorption and bioavailability need, therefore, to be investigated and Caco-2 cell culture model appeared as a promising tool. We have shown herein that the addition of a grape seed extract (GSE) to Dulbecco's modified Eagle's medium (DMEM) used for Caco-2 cell culture leads to a substantial loss of catechin, epicatechin and B2 and B3 dimers from GSE in the medium after 24 h and to a production of hydrogen peroxide (H2O2). When 1420 microM ascorbic acid is added to the DMEM, such H2O2 production was prevented. This hydrogen peroxide generation substantially involves inorganic salts from the DMEM. We recommend that ascorbic acid be added to circumvent such a risk.     

The cytosolic and transmembrane domains of the beta 1,6 N-acetylglucosaminyltransferase (C2GnT) function as a cis to medial/Golgi-targeting determinant

The beta 1,6 N-acetylglucosaminyltransferase (C2GnT) has been recently mapped to the cis/medial-Golgi compartment. To analyze the Golgi-targeting determinants of C2GnT, we constructed various deletion mutants of the enzyme fused to the enhanced green fluorescent protein (EGFP) and localized these proteins by fluorescence microscopy in living cells. We found that the N-terminal peptide encompassing amino acids 1 to 32 represents the minimal Golgi-targeting signal sufficient to localize EGFP to the same compartment as the full-length C2GnT. This peptide makes up the cytoplasmic and the transmembrane domains of the enzyme and was referred to as CTd (cytoplasmic and transmembrane domains). We compared the Golgi-targeting efficiency of the C2GnT-derived CTd with its homologous domains from other glycosyltransferases, including the H-type alpha(1,2)-fucosyltransferase (FucTI), the polypeptide N-acetylgalactosaminyltransferase-I (GalNAcT-I), the alpha(1,3)-fucosyltransferase VII (FucTVII), and the alpha(2,6)-sialyltransferase (ST6Gal-I) and found that the Golgi-targeting determinants of these glycosyltransferases were also composed of their cytosolic and transmembrane domains. To investigate whether the CTd of C2GnT could serve as a cis to medial Golgi-specific signal, we tested its ability to mislocalize two late-Golgi acting glycosyltransferases FucTI and FucTVII. We show that fusing the C2GnT-derived CTd with the catalytic domain of FucTVII resulted in a complete mislocalization of the enzyme to the C2GnT compartment, with a parallel alteration of sialyl-Lewis x synthesis and P-selectin binding. The intracellular distribution and activity of FucTI, however, were not affected. Thus, CTds of either early or late-Golgi acting glycosyltransferases represent the Golgi-targeting domains of these enzymes. In addition, we show that C2GnT-derived CTd can function as a cis/medial Golgi-targeting determinant.     

Occurrence of mycobacteria in water treatment lines and in water distribution systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region

Dihydroflavonol reductase (DFR) is a key enzyme involved in anthocyanin biosynthesis and proanthocyanidin synthesis in grape. DFR catalyses the reduction of dihydroflavonols to leucoanthocyanidins in the anthocyanin pathway. The DFR products, the leucoanthocyanidins, are substrates for the next step in the anthocyanin pathway and are also the substrates for the proanthocyanidin pathway. In the present study the promoter of the grape dfr gene was cloned. Analysis of the dfr promoter sequence revealed the existence of several putative DNA binding motifs. The dfr promoter was fused to the uidA gene and the control of this fusion and the endogenous dfr gene expression, was studied in transformed plants and in red cell suspension originated from fruits. The dfr promoter-uidA gene fusion was expressed in leaves, roots and stems. Deletions of the dfr promoter influenced the specificity of the expression of the GUS gene fusion in plantlet roots and the level of expression in plants and in the red cell suspension originated from fruits. The deletion analysis of the dfr promoter suggests that a specific sequence located between -725 to -233 might be involved in expression of the dfr gene in fruits. Light, calcium and sucrose induced the dfr gene expression. In the transformed suspension cultures, expression of both the endogenous dfr gene and the dfr promoter-uidA gene fusions was induced by white light. The induction by both light and calcium suggests the possible involvement of a UV receptors signal transduction pathway in the induction of the dfr gene. The induction of the dfr gene and the dfr promoter-uidA gene fusions by light and sucrose indicates a close interaction between sucrose and light signalling pathways.     

Reversibility of n-3 fatty acid deficiency-induced changes in dopaminergic neurotransmission in rats: critical role of developmental stage

Previous investigations have shown that the lipid composition of cerebral membranes and dopaminergic neurotransmission are changed under chronic alpha-linolenic acid diet deficiency in the rat. This study investigated whether these changes could be reversed and if the stage of brain maturation might play a role in the recovery process. The effects of reversion on the fatty acid (FA) composition and dopaminergic neurotransmission were studied in brain regions known to be affected by such deficiency (i.e., the prefrontal cortex and nucleus accumbens) in 2-month-old animals. Dopamine release under pharmacological stimulation was studied using a dual-probe microdialysis method. Vesicular monoamine transporters were studied using quantitative autoradiography. The reversal diet, with adequate levels of n-6 and n-3 polyunsaturated fatty acids (PUFAs), was given to deficient rats at different stages of development (0, 7, 14, or 21 days of age). The results showed that when given during the lactating period, this diet was able to restore both the FA composition of brain membranes and the parameters of dopaminergic neurotransmission studied. However, when given from weaning, it allowed partial recovery of biochemical parameters but no recovery of neurochemical factors. The occurrence of profound n-3 PUFA deficiency during the lactating period could therefore be an environmental insult leading to irreversible damage to specific brain functions.     

Proinflammatory gene induction by platelet-activating factor mediated via its cognate nuclear receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H(2)O(2). Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-kappaB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.     

A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.     

Characterization of ThOX proteins as components of the thyroid H(2)O(2)-generating system

We have recently cloned two thyroid-specific cDNAs encoding new members of the NADPH oxidase family. ThOX1 and ThOX2 proteins are colocalized with thyroperoxidase at the apical membrane of human thyroid cells. In the present study we have determined their subcellular localization and maturation in relation to their enzymatic activity. A majority of ThOX proteins accumulated inside the cell and only a small fraction was expressed at the surface. Western blots demonstrated that ThOX's are glycoproteins of 180,000 and 190,000. When totally deglycosylated the molecular weight of both ThOX1 and ThOX2 drops to 160,000. Ca(2+) stimulates the basal H(2)O(2) generation in PC Cl3 cells at a level corresponding to 20% of the leukocyte H(2)O(2) production stimulated by PMA. Nonthyroid cell lines transfected with ThOX1 and ThOX2 show only a single immunoreactive band in Western blot analysis, corresponding to the protein of 180,000. This "immature" protein remains exclusively intracellular and does not present any enzymatic activity. This is not modified by coexpression of thyroperoxidase and p22(Phox). Transfection of ThOX cDNAs into PLB-XCGD cells does not reconstitute their NADPH oxidase activity. We conclude that (1) the thyroid contains some elements of the leukocyte H(2)O(2)-generating system but not all of them; (2) ThOX's are predominantly or exclusively located inside the cell in thyrocytes or in transfected cells, respectively, and as such they are inactive; (3) ThOX's cannot replace gp91(Phox) in the leukocyte; and (4) the thyroid H(2)O(2)-generating system is analogous to the leukocyte system with regard to ThOX's and gp91(Phox) but very different in other aspects. Additional thyroid-specific components are probably required to get complete protein processing and full enzymatic activity in the thyroid.