Human inter-alpha-inhibitor family in acute inflammation: simultaneous synthesis of positive and negative acute-phase proteins.
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Acute-Phase Proteins↗
Biomedical subjects
P Rouet
Publications and source records attributed to P Rouet.
Optimized assays for quantifying transient expressions of co-transfected beta-galactosidase and CAT reporter genes.
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Automation↗
Synergistic induction of interleukin-6 production and gene expression in human thymic epithelial cells by LPS and cytokines.
We examined the ability of LPS and several cytokines (TNF-alpha, IL-1-beta, IFN-gamma, IL-4) to modulate IL-6 production by cultured human thymic epithelial cells (TEC). IL-6 activity was measured by the hybridoma growth factor biological activity. Moderate but detectable IL-6 activity was spontaneously produced in the presence of serum proteins. LPS as well as the cytokines TNF-alpha and IL-1-beta was a potent inducer of IL-6, increasing, respectively, IL-6 levels by 9-, 28-, and 75-fold (mean values) while IL-4 and IFN-gamma provoked no significant effect. Interestingly, clearly different kinetics were observed for IL-6 induction by the various activation agents, the maximal effect being reached at 24, 48, and 72 hr, respectively for LPS, TNF-alpha, and IL-1-beta. Moreover, a synergistic effect of TNF-alpha and either LPS or IL-1-beta was observed. Indeed, TEC incubated with the cytokines in combination at optimal doses produced 5- to 170-fold more IL-6 than TEC stimulated with the cytokines individually. Neutralizing anti-IL-6 polyclonal and monoclonal antibodies completely blocked hybridoma proliferation stimulating activity of TEC supernatants; thus, implying that this activity is essentially due to IL-6. In situ hybridization analysis of cytocentrifuged TEC with an mRNA antisense probe specific for human IL-6 and labeled with 35S demonstrated that up to 90% of TEC could be induced to express the IL-6 gene. Computer-aided quantification of IL-6 mRNA levels indicated that upon stimulation with TNF-alpha combined to LPS, both the numbers of cells expressing IL-6 mRNA and the amounts of cytoplasmic IL-6 mRNA per cell were increased. Taken altogether these results demonstrate that LPS and/or cytokines can modulate and synergistically stimulate IL-6 production. In addition to a possible role in regulating normal thymic T cell activation, the IL-6 produced by TEC could be of pathophysiological relevance in disregulated situations such as in hyperplastic thymuses from patients with myasthenia gravis.
Child, Preschool↗
Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis.
Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1. All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.
Chromosome Mapping↗
Purification and comparison of liver microsomal flavin-containing monooxygenase from normal and streptozotocin-diabetic rats.
Liver microsomal flavin-containing monooxygenase (FMO) activity towards thiobenzamide is two-fold increased in streptozotocin diabetic (insulin deficient) rats and mice and, to a lesser degree in congenital insulin resistant Ob/Ob mice. No difference in thermal stability appears between microsomal FMOs from both normal and diabetic rats. FMO has been purified to homogeneity from these two sources, with a 50-fold increase of specific activity. Their apparent molecular weight is respectively 50,000 and 49,000 and a discrete modification appears in the HPLC profiles of tryptic peptides from purified FMOs. They appear immunochemically very similar and present in equal quantity in microsomal membranes from both normal and diabetic rats, so that the increased activity cannot be ascribed to an increased concentration of the enzyme protein.
Animals↗
Aldrin epoxidase activity in liver microsomes from normal or streptozotocin-diabetic rats: comparison with activity in isolated hepatocytes from normal rats incubated with glucagon.
Aldrin epoxidase activity in liver microsomes from streptozotocin-diabetic rats is only 40% of that from normal rats. Epoxidation of aldrin has also been assayed in freshly isolated hepatocytes from normal rats. Addition of 10(-7) M glucagon to the incubation medium leads to a decreased aldrin epoxidase activity. Owing to the previously reported phosphorylation of a purified cytochrome P-450 isozyme, it is postulated that the cytochrome P-450 dependent aldrin epoxidase may be regulated by a glucagon induced phosphorylation process.
Animals↗
Possible role for thymine glycol in the selective inhibition of DNA synthesis on oxidized DNA templates.
Single-stranded DNA of coliphage M13mp8 was treated with the oxidizing agent, KMnO4, under conditions that selectively form cis-5,6-dihydro-5,6-dihydroxythymine (thymine glycol). Treatment of DNA with 0.7 and 1.4 mM KMnO4 introduced approximately 200 and 400 thymine glycol residues, respectively, per genome. When these DNAs were used to transform Escherichia coli, it was observed that phage survival was reduced in a dose-dependent manner. In studies designed to investigate the effect of DNA oxidation products on replication in vitro, a complementary 15-mer oligodeoxynucleotide was annealed to the oxidized template and extended with the Klenow fragment of DNA polymerase I from E. coli. It was observed that lesions in oxidized DNA strongly inhibited DNA elongation and that DNA synthesis was stopped opposite thymine residues. This is taken as suggestive evidence that the thymine glycol is inhibitory to DNA replication.
Coliphages↗
Metabolism of benzo[a]pyrene by mouse brain microsomes during perinatal development.
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Animals↗
Ontogeny of benzo(a)pyrene hydroxylase, epoxide hydrolase and glutathione-S transferase in the brain, lung and liver of C57Bl/6 mice.
Typical developmental patterns of three drug-metabolizing enzymes in C57Bl/6 mouse brain, lung and liver could be divided into three stages: stage I, at the end of intrauterine life, where an increase in enzymatic activity was observed; stage II, during the first days after birth, where a decrease was seen; and stage III from the 6th day until weaning, where there was a gradual increase, reaching the same values as in the adult animal. However, pulmonary benzo(a)pyrene hydroxylase activity showed an abrupt burst starting on day 6 of postnatal life, then decreased slowly to become steady, and finally increased again. Our data show that the major metabolic pathways catalyzed by glutathione-S transferase and epoxide hydrolase are operative in mouse fetal brain and lung, just as in liver. In addition, we show that enzymatic systems are inducible during fetal life by exogenous compounds such as 5,6-benzoflavone. The early developmental evolution of the drug-metabolizing enzymes should help understand feto-toxicological and teratological phenomena.
Animals↗
Endogenous induction of epoxide hydrolase, benzo(a)pyrene hydroxylase and glutathione-S-transferase in "responsive" C57Bl/6 mice and in "nonresponsive" DBA/2 mice during pregnancy.
Gestational changes in activity for three enzymes associated with different hepatic and pulmonary drug metabolizing systems were investigated in C57Bl/6 and DBA/2 mice: benzo(a)pyrene hydroxylase, epoxide hydrolase and glutathione-S-transferase. The gestational profiles of hepatic and pulmonary benzo(a)pyrene hydrolase, and epoxide hydrolase were similar in both strains. In addition, we demonstrated higher endogenous stimulation of the three studied enzymic activities in C57Bl/6 mice. During the second moiety of pregnancy, temporal variations were observed: a peak of activity occurred between days 17 and 18 for lung and liver benzo(a)pyrene hydroxylase and at day 20 for epoxide hydrolase in both strains. Hepatic glutathione-S-transferase variations were similar in both strains. However, pulmonary glutathione-S-transferase increased gradually throughout pregnancy in C57Bl/6 mice, while a peak of glutathione-S-transferase activity occurred on day 18 of gestation in DBA/2 mice.
Animals↗
Metabolism of benzo[a]pyrene by brain microsomes of fetal and adult rats and mice. Induction by 5,6 benzoflavone, comparison with liver and lung microsomal activities.
Using brain, lung and liver microsomes as the enzyme source in in vitro assays, benzo[a]pyrene (B[a]P) metabolism was studied in fetuses and dams of mice (C57B1/6) and rats (WAG). Separation and quantitation of B[a]P metabolites were performed by h.p.l.c. Microsomal preparations were tested for cytochrome P-450 dependent O-dealkylation of 7-ethoxycoumarin and epoxide hydrolase activities. Another parameter measured included the conjugation of 1-chloro-2,4 dinitrobenzene to glutathione by cytosolic glutathione-S-transferase activity. The induction of B[a]P metabolism was studied after treatment of animals with 5,6-benzoflavone (BF). Mixed function oxygenase, epoxide hydrolase and glutathione-S-transferase activities were transplacentally inducible after dams were treated with BF. Metabolic activation of B[a]P by fetal brain microsomes was lower in both species than that by fetal lung and liver microsomes, but it was higher in fetuses than in adults. All metabolites of B[a]P increased after BF treatment; the production of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene (7,8-dihydrodiol B[a]P) was higher in brain microsomes from BF-treated rats than that in mice. In stimulated rats, the formation of 7,8-dihydrodiol B[a]P by fetal brain microsomes were higher than that by fetal lung microsomes, whereas in mice, the opposite was observed. These data suggest that initiation could occur in utero, and partially explain the species-specific differences in susceptibility to transplacental tumorigenesis by polycyclic aromatic hydrocarbons by differences in biotransformation in the target organ.
Animals↗
[Study of ventricular repolarization in an experimental model of arterial hypertension associated with obesity].
UNLABELLED: High fat diet (HFD) in dogs is associated with obesity and hypertension (HTN) but also with a significant and early decrease in heart rate variability (HRV). Decreased HRV has been shown to be a good predictor of sudden cardiac death due mainly to arrhythmic event. The aim of this work was to investigate the changes in ventricular repolarization through 24 hours EKG recordings in dogs with hypertension and rendered obese by 20 weeks of HFD. This was achieved through 24 hour EKG recording analysis of QT parameters. The aims of this work was i) feasibility of this method in dogs and ii) identification of potential arrhythmic risk factors that could explain overmortality during obesity. METHOD: Six dogs received a high fat diet (HFD) ad libitum during 20 weeks. A 24 hour EKG recording was realized just before and after 20 weeks of HFD. The following parameters studying QT interval were collected: QT interval lasting from the beginning of the Q wave to the apex (QTa) and to the end of the T wave (QTe), QT intervals plotted against RR intervals and two regression lines were calculated characterized by their slope and intersection with the Y axis, QT dispersion (longest minus shortest QT interval for each RR value) as well as the difference of QT interval between night and day at a fixed RR value considered as a marker of the sympathovagal balance. Our results show that HFD significantly increased body weight, blood pressure, heart rate, left ventricular mass and insulinemia. QT dispersion was increased in a non-significant manner both during day (+35%) and night (16%) for QTa and only during day for QTe (+27%). This increased dispersion of QT was not associated to any increase of QT interval. There was no effect of HFD on QT dynamicity parameter nor on the night-day difference at any RR interval from 300 to 1,300 ms. CONCLUSION: HFD tend increase QT dispersion without any effect on QT interval. These results are compatible with a heterogeneous repolarization probably related to abnormal autonomic nervous system tone. This study could partly explain occurrence of lethal arrhythmias during obesity which might lead to overmortality of obese patients. These results are different for QTa and QTe, but these two parameters are characterizing different type of ventricular cells. This study confirms the feasibility of this method in an experimental model, but results need to be validated in larger groups and in human.
Animals↗
[Early atrial gene regulation of obesity-related arterial hypertension].
High fat diet (HFD) induces both arterial hypertension and tachycardia in dogs. Changes in heart rate occur early and are in part due to a decrease in the parasympathetic drive to the heart secondary to down-regulation of atrial muscarinic M2 receptors (Pelat et al. Hypertension 1999; 340: 1066-72). These data suggest that HFD is able to modify genic expression at atrial level. Thus, the aim of this work was to perform a systematic study of the genic expression profile in dogs made obese and hypertensive by 9 weeks of HFD. Blood pressure and heart rate were measured by telemetry implanted 15 days before starting regimen in 6 HFD and in 6 control dogs. HFD was the normal canine diet administered to controls but mixed with 300 g of beef fat. At the end of the experience, animals were sacrified and right atria were collected. Gene regulation was assessed in pooled tissue samples from both groups using suppressive substractive hybridization and microarray analysis. Genes with induction or repression rates of at least 20% when compared to controls were sequenced. As previously reported HFD induced a significant increase in body weight, blood pressure and heart rate when compared to controls. The results of SSH experiments led to the identification of 32 genes which are differentially regulated in atria from HFD dogs. Most are genes encoding proteins which have been previously shown to be regulated during various cardiopathies (MMP9, Na/K-ATPase 3...). These changes indicate the existence of early remodeling processes of atrial myocardium secondary to HFD. Other group of genes encodes proteins with no role identified in heart up today (lec-3, ERK-3, TRIP1, nucleophosmin...) or which function remains totally unknown. This work confirms that HFD is associated with early changes in gene expression in atrium. These changes are unlikely to be related to ventricular hypertrophy which is observed only during long-term HFD. Further studies are necessary to demonstrate the role of these modifications in the pathophysiological mechanisms leading to the increase in heart rate in this model of obesity-related arterial hypertension.
Animals↗
Effects of genetic or chemically induced diabetes on imipramine metabolism. Respective involvement of flavin monooxygenase and cytochrome P-450-dependent monooxygenases.
Imipramine metabolism has been studied in both type I (streptozotocin-induced insulin-deficient) and type II (genetically insulin-resistant) diabetes in mice. In both types of diabetes, the formation of imipramine N-oxide is increased. In type I diabetes, desmethyl- and 2-hydroxyimipramine are additionally increased. The inhibition of imipramine metabolism by anti-cytochrome P-450 reductase antibodies led to the conclusion that cytochrome P-450-dependent monooxygenases are not involved in the N-oxidation of imipramine. This metabolic route is only supported by the flavin monooxygenase, whose activity is increased by diabetes. The pharmacological implications of altered imipramine metabolism in diabetic states are discussed in relation to the drug metabolism in human diabetes.
Animals↗