Documenting a time-bound, circular view of hierarchies: a microanalysis of parent-infant dyadic interaction.

This article presents a new theory that separates the levels of communication and relates them circularly, namely, by separating time from space/meaning variables. Documenting this proposition requires sequential microdescriptions--a far-out project in the field of family therapy. In an extensive study of clinical and nonclinical families, starting with available microanalytic data on nonverbal parent-infant dialogue, distinct time organizations have been found to modify the degree of circularity between the levels of interaction according to the observed types of engagement, that is, consensual, conflictual, and paradoxical. The double description of the dyad as a totality versus the dyad as a framing/developing organization imparts crucial information on how development proceeds in dyadic, co-evolutive systems, and presumably in larger ones too. In this perspective, a model is elaborated and then applied to a case description in our therapeutic consultation.

Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

Differential expression of triiodothyronine receptors in schwannoma and neurofibroma: role of Schwann cell-axon interaction.

Regulation of gene expression in Schwann cells may be determined, at least in part, by the interaction of these cells with axons. Two peripheral nerve tumors, neurofibroma and schwannoma, represent good tools for studying Schwann cell activity in the presence or absence of axon action. In the present work we studied the expression of triiodothyronine receptors (T3R) by Schwann cells in these two tumors and also in adult normal sciatic nerve. Confirming the results of the histological examination, immunostaining of the neurofilaments showed the presence of fascicles or scattered axons in all neurofibroma sections studied. In these neurofibromas, Schwann cells did not express T3R immunoreactivity. Furthermore, in adult normal sciatic nerve, Schwann cells which ensheathed axons were devoid of any T3R expression. In contrast, in schwannoma, the complete absence of axons was demonstrated by the lack of neurofilament immunostaining. Here, Schwann cells deprived of axonal interaction displayed clear T3R immunoreactivity. In schwannoma cell cultures, Schwann cells continued to express T3R, even in cultures treated with medium that had been conditioned with rat sensory neurons. On the basis of these results, we suggest that, beside the possible regulatory mechanisms for T3R, the synthesis of T3R is regulated, at least in part, by Schwann cell-axon interaction.

Pharmacokinetic fluvoxamine-clomipramine interaction with favorable therapeutic consequences in therapy-resistant depressive patient.

We describe the case of a depressive patient who was a rapid metabolizer of CYP2D6 substrates and a heavy smoker, and who did not respond to several courses of treatment with antidepressants, as a result of unusually low drug-plasma levels. During hospitalization, he did not improve after treatment with clomipramine (150-225 mg/day during three weeks), but showed a response within four days after addition of fluvoxamine (100 mg/day). Plasma levels of clomipramine and desmethylclomipramine changed from 58 ng/ml and 87 ng/ml to 223 ng/ml and 49 ng/ml respectively one week after addition of fluvoxamine. Present knowledge of the role of cytochrome P-450 isozymes, such as CYP1A2, CYP2C19, CYP2D6, and CYP3A4, in the metabolism of psychotropic drugs as well as therapeutic drug-plasma level monitoring may thus help to determine individual treatment.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Interaction between dietary lipids an physical inactivity on insulin sensitivity and on intramyocellular lipids in healthy men

Résumé Interaction entre les lipides alimentaires et l'inactivité physique sur la sensibilité à l'insuline et les lipides intramyocellulaires chez le sujet masculin en bonne santé Ces deux dernières décennies, l'incidence de la résistance à l'insuline n'a cessé de progresser dans les pays industrialisés. Un grand nombre de travaux suggèrent que ce trouble métabolique joue un rôle important dans la pathogenèse de maladies propres au monde industrialisé, telles que le diabète, l'hypertension et les maladies cardiovasculaires. Malgré de nombreuses études, les mécanismes à l'origine de la résistance à l'insuline restent encore incomplètement élucidés. En plus d'une composante génétique, de nombreux facteurs environnementaux semblent impliqués parmi ces derniers, nous nous sommes intéressés à l'effet d'une alimentation riche en graisses associée à une période d'inactivité physique de courte durée. Nous nous sommes également penchés sur la corrélation décrite entre la résistance à l'insuline et la concentration de graisses présentes à l'intérieur des cellules musculaires squelettiques, appelées lipides intramyocellulaires. Pour ce faire, 8 volontaires masculins ont été étudiés à deux occasions. Après deux jours de diète équilibrée associée à une activité physique, les participants étaient confinés au lit strict pour 60 heures et devaient manger une alimentation soit riche en graisses saturées soit riche en hydrates de carbones. Pour évaluer l'effet de l'alimentation seule, 6 des 8 volontaires ont été réétudiés après deux jours de diète équilibrée suivie par 60 heures d'alimentation riche en graisses saturées associées à une activité physique contrôlée. Nous avons estimé la sensibilité à l'insuline par la technique du clamp hyperinsulinémique euglycémique alors que la concentration de lipides intramyocellulaires a été déterminée par spectroscopie par résonance magnétique. Après 60 heures d'inactivité physique associée à une alimentation riche en lipides, nous avons observé une diminution de l'utilisation de glucose dépendante de l'insuline (-24±6%; p<0.05), alors qu'aucune modification significative de ce même paramètre n'a été constatée lorsque l'inactivité physique était associée à une alimentation riche en hydrates de carbones (+19±10%). Ces deux conditions se sont accompagnées d'une augmentation des lipides intramyocellulaires (+32±7% et +17±8% respectivement). Bien que l'augmentation des lipides intramyocellulaires observée après 60 heures d'une alimentation riche en graisses saturées associée à une activité physique modérée fût d'une ampleur similaire à celle de la condition associant une alimentation riche en graisses et inactivité physique, l'utilisation de glucose induite par l'insuline n'a pas été modifiée de manière significative (-7±9%) Ces résultats indiquent que l'inactivité physique et une alimentation riche en graisses saturées semblent interagir, induisant une diminution de la sensibilité à l'insuline globale. La concentration de lipides intramyocellulaires a été influencée par les lipides issus de l'alimentation et l'inactivité physique, sans être toutefois corrélée à la résistance à l'insuline. Abstract OBJECTIVE - To assess the effect of a possible interaction between dietary fat and physical inactivity on whole-body insulin sensitivity and intramyocellular lipids (IMCLs). RESEARCH DESIGN AND METHODS - Eight healthy male volunteers were studied on two occasions. After 2 days of an equilibrated diet and moderate physical activity, participants remained inactive (bed rest) for 60 h and consumed either a high-saturated fat (45% fat, of which ~60% was saturated fat [BR-HF]) or a high-carbohydrate (70% carbohydrate [BR-HCHO]) diet. To evaluate the effect of a high-fat diet alone, six of the eight volunteers were restudied after a 2-day equilibrated diet followed by 60 h on a high-saturated fat diet and controlled physical activity (PA-HF). Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp and IMCL concentrations by H-magnetic resonance spectroscopy. RESULTS - Insulin-mediated glucose disposal was decreased by BR-HF condition (-24 ± 6%, P < 0.05) but did not change with BR-HCHO ( + 19 ± 10%, NS). BR-HF and BR-HCHO increased IMCL levels (+32 ± 7%, P < 0.05 and +17 ± 8%, P < 0.0011, respectively). Although the increase in IMCL levels with PA-HF (+31 ± 19%, P = 0.12) was similar to that during BR-HF, insulin-mediated glucose disposal ( -7 ± 9%, NS) was not decreased. CONCLUSIONS - These data indicate that physical inactivity and a high-saturated fat diet may interact to reduce whole-body insulin sensitivity. IMCL content was influenced by dietary lipid and physical inactivity but was not directly associated with insulin resistance.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Dissecting TCR-MHC/peptide complex interactions with A2/peptide multimers incorporating tumor antigen peptide variants: crucial role of interaction kinetics on functional outcomes.

Soluble peptide/MHC-class-I (pMHC) multimers have recently emerged as unique reagents for the study of specific interactions between the pMHC complex and the TCR. Here, we assessed the relative binding efficiency of a panel of multimers incorporating single-alanine-substituted variants of the tumor-antigen-derived peptide MAGE-A10(254-262) to specific CTL clones displaying different functional avidity. For each individual clone, the efficiency of binding of multimers incorporating MAGE-A10 peptide variants was, in most cases, in good although not linear correlation with the avidity of recognition of the corresponding variant. In addition, we observed two types of discrepancies between efficiency of recognition and multimer binding. First, for some peptide variants, efficient multimer binding was detected in the absence of measurable effector functions. Some of these peptide variants displayed antagonist activity. Second, when comparing different clones we found clear discrepancies between the dose of peptide required to obtain half-maximal lysis in CTL assays and the binding efficiency of the corresponding multimers. These discrepancies, however, were resolved when the differential stability of the TCR/pMHC complexes was determined. For individual clones, decreased recognition correlated with increased TCR/pMHC off-rate. TCR/pMHC complexes formed by antagonist ligands displayed off-rates faster than those of TCR/pMHC complexes formed with weak agonists. In addition, when comparing different clones, the efficiency of multimer staining correlated better with relative multimer off-rates than with half-maximal lysis values. Altogether, the data presented here reconcile and extend our previous results on the impact of the kinetics of interaction of TCR with pMHC complexes on multimer binding and underline the crucial role of TCR/pMHC off-rates for the functional outcome of such interactions.

Remediation of a marine shore tailings deposit and the importance of water-rock interaction on element cycling in the coastal aquifer

We present the study of the geochemical processes associated with the first successful remediation of a marine shore tailings deposit in a coastal desert environment (Bahia de Ite, in the Atacama Desert of Peru). The remediation approach implemented a wetland on top of the oxidized tailings. The site is characterized by a high hydrauliz gradient produced by agricultural irrigation on upstream gravel terraces that pushed river water (similar to 500 mg/L SO(4)) toward the sea and through the tailings deposit. The geochemical and isotopic (delta(2)H(water) and delta(18)O(water), delta(34)S(sulfate) , delta(18)O(sulfate)) approach applied here revealed that evaporite horizons (anhydrite and halite) in the gravel terraces are the source of increased concentrations of SO(4), Cl, and Na up to similar to 1500 mg/L in the springs at the base of the gravel terraces. Deeper groundwater interacting with underlying marine sequences increased the concentrations of SO(4), Cl, and Na up to 6000 mg/L and increased the alkalinity up to 923 mg/L CaCO(3) eq. in the coastal aquifer. These waters infiltrated into the tailings deposit at the shelf-tailings interface. Nonremediated tailings had a low-pH oxidation zone (pH 1-4) with significant accumulations of efflorescent salts (10-20 cm thick) at the surface because of upward capillary transport of metal cations in the arid climate. Remediated tailings were characterized by neutral pH and reducing conditions (pH similar to 7, Eh similar to 100 mV). As a result, most bivalent metals such as Cu, Zn, and Ni had very low concentrations (around 0.01 mg/L or below detection limit) because of reduction and sorption processes. In contrast, these reducing conditions increased the mobility of iron from two sources in this system: (1) The originally Fe(III)-rich oxidation zone, where Fe(II) was reduced during the remediation process and formed an Fe(II) plume, and (2) reductive dissolution of Fe(III) oxides present in the original shelf lithology formed an Fe-Mn plume at 10-m depth. These two Fe-rich plumes were pushed toward the shoreline where more oxidizing and higher pH conditions triggered the precipitation of Fe(HI)hydroxide coatings on silicates. These coatings acted as a filter for the arsenic, which naturally infiltrated with the river water (similar to 500 mu g/L As natural background) into the tailings deposit.

Identification of key amino acids of the mouse mammary tumor virus superantigen involved in the specific interaction with T-cell receptor V(beta) domains.

Mouse mammary tumor virus (MMTV) is a retrovirus encoding a superantigen that is recognized in association with major histocompatibility complex class II by the variable region of the beta chain (V(beta)) of the T-cell receptor. The C-terminal 30 to 40 amino acids of the superantigen of different MMTVs display high sequence variability that correlates with the recognition of particular T-cell receptor V(beta) chains. Interestingly, MMTV(SIM) and mtv-8 superantigens are highly homologous but have nonoverlapping T-cell receptor V(beta) specificities. To determine the importance of these few differences for specific V(beta) interaction, we studied superantigen responses in mice to chimeric and mutant MMTV(SIM) and mtv-8 superantigens expressed by recombinant vaccinia viruses. We show that only a few changes (two to six residues) within the C terminus are necessary to modify superantigen recognition by specific V(beta)s. Thus, the introduction of the MMTV(SIM) residues 314-315 into the mtv-8 superantigen greatly decreased its V(beta)12 reactivity without gain of MMTV(SIM)-specific function. The introduction of MMTV(SIM)-specific residues 289 to 295, however, induced a recognition pattern that was a mixture of MMTV(SIM)- and mtv-8-specific V(beta) reactivities: both weak MMTV(SIM)-specific V(beta)4 and full mtv-8-specific V(beta)11 recognition were observed while V(beta)12 interaction was lost. The combination of the two MMTV(SIM)-specific regions in the mtv-8 superantigen established normal MMTV(SIM)-specific V(beta)4 reactivity and completely abolished mtv-8-specific V(beta)5, -11, and -12 interactions. These new functional superantigens with mixed V(beta) recognition patterns allowed us to precisely delineate sites relevant for molecular interactions between the SIM or mtv-8 superantigen and the T-cell receptor V(beta) domain within the 30 C-terminal residues of the viral superantigen.