On the DNA cleavage mechanism of Type I restriction enzymes

Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5′- and 3′-overhangs of varying lengths. EcoAI preferentially generated 3′-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5′-overhangs of a length of ~6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.     

Inter-modal learning task in bottlenosed dolphins (Tursiops truncatus): a preliminary study showed that social factors might influence learning strategies

We studied the dolphins capacity to perform a cognitive task when information arrives through two different sensory modalities (visual and auditory). Three female bottlenosed dolphins (Tursiops truncatus) were submitted to a two-, three-, or four-choice visually combined with auditory discrimination problem without any food reward. Since they were not isolated, we examined the significance of the role of identified social constraints on the learning process. The results tended to show an ability to associate simple visual forms and auditory information using an underwater touchscreen. This task was learnt in various ways according to identified social constraints. For each subject, we examined and discussed the number of successful answers, their ability to focus attention on the test and to acquire, or not, the specific task, while noting influences of the social interactions and the social hierarchy on the whole acquisition process.     

Identification of two tamoxifen target proteins by photolabeling with 4-(2-morpholinoethoxy)benzophenone

Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.     

Molecular phylogeny of parabasalids inferred from small subunit rRNA sequences,with emphasis on the Devescovinidae and Calonymphidae (Trichomonadea)

Small subunit rRNA sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the polymastigont Calonymphidae, including Snyderella tabogae, Calonympha grassii, and Metacoronympha senta. The yet-unidentified sequence Nk9 previously obtained from the termite Neotermes koshunensis, has also been shown to derive from the Devescovinidae Devescovina sp. by in situ hybridization. These new sequences were analyzed by distance, parsimony, and likelihood methods in a broad phylogeny including all identified parabasalid sequences available in databases. All analyses revealed the emergence of a very well supported Devescovinidae/Calonymphidae group but showed an unexpected dichotomy of the Calonymphidae represented by the "Coronympha" and "Calonympha" groups. It strongly suggests that the polymastigont state observed in the Calonymphidae might be explained by at least two independent evolutionary events. In a second phylogenetic analysis, some yet-unidentified parabasalid sequences likely deriving from the Devescovinidae/Calonymphidae taxa, were added to our data set. This analysis confirmed the polyphyly of the Calonymphidae. A tentative identification is proposed for each of these sequences, and hypotheses on the origin of the Devescovinidae and Calonymphidae are discussed. Tritrichomonas foetus or a close relative might be the best candidate for the ancestor of the Devescovinidae, fairly consistent with morphology-based hypotheses. Regarding the Calonymphidae, the origin of the "Coronympha" group might be found within the Devescovinidae, related to Foaina, whereas the "Calonympha" group may directly descend from Tritrichomonas or related species.     

Antigen-experienced T cells undergo a transient phase of unresponsiveness following optimal stimulation

Interaction of the Ag-specific receptor of T lymphocytes with its Ag/MHC ligand can lead either to cell activation or to a state of unresponsiveness often referred to as anergy. It has been generally assumed that anergy develops as a consequence of inadequate stimulation, such as in response to altered peptide ligands or to agonists presented by costimulatory-deficient accessory cells. The present study uncovers an alternative way of inducing an unresponsive state in T cells. Indeed, we demonstrate herein that Ag-stimulation of murine CD4+ Th clones induces cellular activation, characterized by cytokine production and cell proliferation, followed by a state of transient (lasting up to 6 days) unresponsiveness to further antigenic stimulation. This state of activation-induced unresponsiveness 1) is not a consequence of inadequate costimulation, as it occurs when cells are stimulated in the presence of dendritic cells or anti-CD28 Abs; 2) develops after an optimal response to Ag; 3) is not due to cell death/apoptosis or CTLA-4 engagement; 4) down-regulates the proliferation and cytokine production of both Th1- and Th2-like clones; and 5) does not affect the early steps of signal transduction. Finally, naive T cells are not sensitive to this novel form of unresponsiveness, but become gradually susceptible to activation-induced unresponsiveness upon Ag stimulation. Collectively, these data suggest that activation-induced T cell unresponsiveness may represent a regulatory mechanism limiting the clonal expansion and effector cell function of Ag-experienced T cells, thus contributing to the homeostasis of an immune response.     

Effects of amiodarone on cardiac function and mitochondrial oxidative phosphorylation during ischemia and reperfusion

This study was carried out in order to determine if the efficiency of amiodarone, a class III antiarrhythmic agent, is associated with changes in mitochondrial oxidative phosphorylation. A population of 30 rats were treated with amiodarone (100 mg/kg/day) for 5 days. A second population receiving only vehicle was used as control. The hearts were perfused according to the working mode. After 15 min of normoxic perfusion, the left main coronary artery was ligated and the ligation was maintained for 20 min. The ligation was removed and reperfusion continued for a further 30 min. The electrocardiogram was monitored continuously. At the end of perfusion, the ischemic and non ischemic areas were visually separated and mitochondria were harvested from each area. Their oxidative and energy metabolism were assessed with palmitoylcarnitine as substrate in 2 respiration media differing in their free calcium concentration (0 or 0.34 m). In normoxic conditions, amiodarone treatment increased the cardiac metabolic efficiency (mechanical work to oxygen consumption ratio). The local ischemia decreased the aortic and coronary flows without modifying the cardiac metabolic efficiency. Amiodarone treatment maintained the aortic flow at a significantly higher value; the duration of severe arrhythmias was significantly decreased by the drug. The reperfusion of the ischemic area allowed the partial recovery of fluid dynamics. The coronary flow was restored to 89% of the pre ischemic value. Conversely, the aortic flow never exceeded that measured at the end of ischemia, partly due to the important development of severe arrhythmias. The recovery of aortic flow and metabolic efficiency during reperfusion was improved by amiodarone treatment; ventricular tachycardia and fibrillation duration were reduced. In the mitochondria issued from the normoxic area, the energy metabolism was not altered by the amiodarone treatment, but the presence of calcium in the respiration medium modified the oxidative phosphorylation. The divalent cation slightly decreased the state III respiration rate and increased noticeably the state IV respiration rate. This was associated with an important mitochondrial AMP production and maintenance of ADP in the respiration medium. This energy wasting was reported to decrease the mitochondrial metabolic efficiency. After an ischemia-reperfusion sequence, mitochondrial oxidation phosphorylation was reduced and amiodarone treatment amplified this decrease. This was presumably due to an increased mitochondrial calcium accumulation. Thus, the beneficial properties of amiodarone during reperfusion are supposed to be due to a protection against the deleterious effect of excess matrix calcium on mitochondrial energy metabolism.