Glutamate optimizes enzymatic activity under high hydrostatic pressure in Desulfovibrio species: effects on the ubiquitous thioredoxin system.

In piezophilic microorganisms, enzymes are optimized to perform under high hydrostatic pressure. The two major reported mechanisms responsible for such adaptation in bacterial species are changes in amino acids in the protein structure, favoring their activity and stability under high-pressure conditions, and the possible accumulation of micromolecular co-solutes in the cytoplasm. Recently, the accumulation of glutamate in the cytoplasm of piezophilic Desulfovibrio species has been reported under high-pressure growth conditions. In this study, analysis of the effect of glutamate on the enzymatic activity of the thioredoxin reductase/thioredoxin enzymatic complex of either a piezosensitive or a piezophilic microorganism confirms its role as a protective co-solute. Analysis of the thioredoxin structures suggests an adaptation both to the presence of glutamate and to high hydrostatic pressure in the enzyme from the piezophilic strain. Indeed, the presence of large surface pockets could counterbalance the overall compression that occurs at high hydrostatic pressure to maintain enzymatic activity. A lower isoelectric point and a greater dipolar moment than that of thioredoxin from the piezosensitive strain would allow the protein from the piezophilic strain to compensate for the presence of the charged amino acid glutamate to interact with its partner.

[Ethical problems of extreme prematurity: results of a qualitative study with obstetricians and midwives]. / Problèmes éthiques posés par l'extrême prématurité: résultats d'une étude qualitative auprès des obstétriciens et des sages-femmes.

OBJECTIVE: Our objective was to explore the practices, attitudes and feelings of obstetricians and midwives in case of extreme prematurity. POPULATION AND METHODS: A qualitative study was conducted as part of a European Concerted Action (EUROBS) in 1999 and 2000 in three tertiary-care maternity units, located in three cities in the northern, southern and central areas of France respectively. Semi-structured, tape-recorded interviews were conducted and were independently analysed by two different researchers using a content analysis. All full-time obstetricians and half of the full-time midwives were eligible for the study. Overall, 17 obstetricians and 30 midwives participated. RESULTS: Both obstetricians and midwives considered that decision-making in case of very preterm births raised ethical problems concerning the mother and the foetus. Despite some birth weight and gestational age criteria defined in advance, management around delivery appeared to be decided on a case-by-case basis. At birth, the neonatologists made the decisions. They were perceived as more inclined than the obstetrical team to initiate intensive care. If the child was born alive, intensive care was started, knowing that it could be withdrawn later, if appropriate. Parents were sometimes involved in decision-making during pregnancy, less frequently at birth or after birth. DISCUSSION AND CONCLUSION: Compared with obstetricians, midwives tended to have a less favourable perception of the neonatologists' practices, and to deplore the lack of parental information and involvement in decision-making. Decisions about the obstetrical management and resuscitation of extremely preterm infants are essentially always made on a case-by-case basis. Parents are sometimes involved in decision-making. Midwives express serious concerns about the current practices.

Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.

The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.

[Psychological consequences of multiple births]. / Conséquences psychologiques des naissances multiples.

Since the mid 1970s, the number of multiple births has dramatically increased in our country and most European countries. This paper summarizes the psychological consequences of multiple births based on a review of the literature and on our clinical experience. During pregnancy mothers experience great physical problems linked with increased medical risks for themselves and for the children. These risks cause psychological difficulties: hospitalisation and separation from the family, fear of a premature delivery and anxiety for the children. After delivery the children are often hospitalized, which makes the attachment process difficult. The mortality of multiple children is high and mourning for one child creates particular problems for parents who simultaneously face grieving and attachment processes. After hospital discharge, the overload of work mothers experience leads to physical and nervous fatigue, which does not make easier individual relationship with the children. Mothers have a high level of psychological vulnerability and an increased risk of depression. The satisfactory development of each twin or triplet child requires individualized relationship with his/her mother and his/her father. That is how he/she will be able to build his/her identity and future autonomy. It is important to be aware of the problems experienced by the families and to improve the way material help and psychological support are provided to them.

[Difficulties with the follow-up of a cohort of very preterm infants: The EPIPAGE Paris-Petite-Couronne population]. / Difficultés de suivi d'une cohorte d'enfants nés grands prématurés : EPIPAGE Paris-Petite-Couronne.

OBJECTIVES: To describe the characteristics of the children of parents who did not respond to a follow-up questionnaire in a cohort of very preterm infants (EPIPAGE study) and to explore the reasons why they did not complete the questionnaires. METHODS: The non-respondents (N = 176) were the parents who did not return the questionnaires at 1 and 2 years. Contacts were organised by telephone calls and at home visits. RESULTS: The families of the non-respondents presented more socioeconomic difficulties and their children were born more preterm than those followed up without difficulties. The efforts made to contact the non-respondents revealed that their families were often confronted with major adverse socioeconomic difficulties, had severe illnesses and psychological troubles. Half of the children of the non-respondents were finally examined at the 5-year assessment. They showed lower performances at the cognitive development test and a poorer health status than the children of the respondents. CONCLUSION: When the follow-up does not include the initial population in its totality, the socioeconomic difficulties are underestimated and probably also the rate of sequelae associated with very preterm delivery.

Delivery room management of extremely preterm infants: the EPIPAGE-2 study.

OBJECTIVE: To analyse the delivery room management of babies born between 22 and 26 weeks of completed gestational age and to identify the factors associated with the withholding or withdrawal of intensive care. STUDY DESIGN: Population-based cohort study. PATIENTS AND METHODS: Our study population comprised 2145 births between 22 and 26 completed weeks enrolled in the EPIPAGE-2 study, a French cohort of very preterm infants born in 2011. The primary outcome measure was withholding or withdrawal of intensive care in the delivery room. RESULTS: Among infants born alive at 22-23 weeks, intensive care was withheld or withdrawn for >90%. At 24 weeks, resuscitative measures were withheld or withdrawn for 38%, at 25 weeks for 8% and at 26 weeks for 3%. Other factors besides gestational age at birth associated with this withholding or withdrawal for infants born at 24-26 weeks were birth weight <600 g, emergency delivery (within 24 h of the mother's admission) and singleton pregnancy. Although rates of withholding or withdrawal of intensive care varied substantially between maternity units (from 0% to 100%), the variability was primarily explained by differences in distributions of gestational age at birth. CONCLUSIONS: Although gestational age is only one factor predicting survival of preterm infants, practices in France appear to be based primarily on this factor, which thus has direct effects on the survival of extremely preterm infants. The ethical implications of basing life and death decisions only on gestational age before 25 weeks require further examination.

Contact inhibition within hemoglobin S polymer by thiol reagents.

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.

Effects of the alpha 20 mutation on the polymerization of Hb S.

The contribution of the alpha 20 residues in intermolecular contacts present in hemoglobin S fibers was investigated with mixtures of Hb Le Lamentin alpha 2(20)His----Gln beta 2A and of hemoglobin S alpha 2A beta 2(6)Glu----Val and with artificial hybrids alpha 2(20)His----Gln beta 2(6)Glu----Val. This study showed an increased solubility and delay time of polymerization of Hb S in solution only when the mutation at the alpha 20 residue is cis to the beta 6 Val contact. No modification of the polymerization process occurs when the mutation is trans to this beta 6 Val contact. This result is in agreement with the crystal model of Wishner and Love, who showed that one of the two alpha 20 residues of the Hb S tetramer was involved in an axial contact between hemoglobin S molecules in the crystals of Hb S ( Wishner , B.C., Ward, K.B., Lattman , E.E. and Love, W.E. (1975) J. Mol. Biol. 98, 179-194). The present observation is a new illustration of the validity of the crystal model for the structure of the fibers based on pairs of double filaments.

Changes of polymerization and conformation of hemoglobin S induced by thiol reagents.

Thiol reagents, covalently bound to cysteine beta 93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2'-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.

Kinetics of polymerization of hemoglobin S modified by thiol reagents and by oxidation.

The effects of four thiol reagents on the kinetics of polymerization of hemoglobin S have been studied in high phosphate buffer (1.8 M), in the presence (3 mM) or absence of sodium dithionite, depending on the reduction of mixed disulfides of Hb in the presence of this reducing agent. The effect of oxidized forms (methemoglobin) of HbS on the kinetics of aggregation of deoxyHbS was also studied because of the presence of 33% metHbS when HbS was modified by 4-aminophenyl disulfide. In the presence of sodium dithionite, the delay times prior to polymerization of deoxyHbS modified by N-ethylmaleimide, iodoacetamide and 4-aminophenyl disulfide were, respectively, 1.5-, 1.35- and 1.15-times longer than that of native deoxyHbS. The results indicate that the radicals bound to the cysteine beta 93 residue inhibit the contacts in the polymer formation to various extents but do not modify the size of the nuclei.

Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties.

Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.

Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.

The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.

Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.

Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.

Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

Structural modeling of the human erythrocyte bisphosphoglycerate mutase.

Using the crystallographic structure of yeast monophosphoglycerate mutase (MPGM) as a framework we constructed a three-dimensional model of the homologous human erythrocyte bisphosphoglycerate mutase (BPGM). The modeling procedure consisted of substituting 117 amino acid residues and positioning 19 C-terminal residues (unresolved in the X-ray structure) by empirical methods, followed by energy minimization. Among several differences in the active site region the most significant appears to be the replacement of Ser11 in MPGM by Gly in BPGM. The C-terminal segment, which contains mainly basic amino acids, lines the cavity of the active site. The seven amino acid residues, which have been shown to be essential for the three catalytic functions of the human BPGM, interact with the amino acids in the protein core, near the active site. In addition, a cluster of several positively charged residues, particularly arginines, has been identified at the entrance of the active site; this cluster may serve as a secondary binding site for polyanionic substrates or cofactors, as required by a two-binding-site model of the catalytic activities. This model is in agreement with recent studies of an inactive BPGM variant substituent at an Arg position situated in this positively charged cluster. The position of Cys20 in the model constructed suggests that this residue is responsible for inactivation of the enzyme by sulfhydryl reagents. Subunit interfaces have also been constructed for BPGM by analogy with MPGM and suggest that, in addition to the known dimerization of BPGM, tetramerization may occur under certain conditions.

Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.

Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.

The effects of a severe drought on mouflon lamb survival.

The mouflon population of Caroux-Espinouse, southern France, inhabits a highly seasonal area with dry summers. We monitored summer lamb survival during a severe drought in 2003, from early June to late August. The survival of 35 radio-tagged lambs over nine two-week periods was strongly affected by the timing of rainfall. Survival depended on the amount of rainfall recorded at a given 14 day period and in the previous 14-21 day period. Survival was not influenced by the exceptionally high mean daily temperature recorded during some periods. Male lamb survival (0.68) tended to be less than female survival (0.81), although not significantly, possibly because of a low sample size. The high lamb mortality (25.7%) recorded during a four-month period is much higher than previous estimates of first-year mortality (less than 10%). We recommend accounting for climatic variation in summer when studying the population dynamics of ungulates.

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.

Psychological consequences of having triplets: a 4-year follow-up study.

OBJECTIVE: To assess the mental health of mothers of triplets and the quality of relationship with the children 4 years after delivery. DESIGN: Prospective follow-up study from delivery, to 4 years. Assessments at home by a psychologist, using semistructured tape-recorded interviews. SETTING: One maternity hospital in Paris, France. PATIENT(S): Eleven consecutive mothers having delivered triplets between October 1988 and February 1990. All except one had conceived after infertility treatment. MAIN OUTCOME MEASURE(S): Evaluation of the mothers' emotional well-being and level of depression measured by the CES-D Scale (Center for Epidemiologic Studies-Depression Scale). Opinion of the mothers about the quality of the relationship with the children. RESULT(S): All mothers reported emotional distress at 4 years, mainly fatigue and stress. Four mothers had a high score of depression and used psychotropic medication. The relationship with the children and difficulties in coping with their aggressive behavior and conflicts were the main reason for psychological distress. Difficulties had not decreased since the previous assessment at 2 years. Four mothers spontaneously expressed regrets about having triplets. CONCLUSION(S): Patients undergoing infertility treatments should receive adequate information about the long-term psychological "cost" of a triplet birth. Infertility treatments should be adapted in order to decrease the risk of triplet pregnancies.

Relation of early neuromotor and cranial signs with neuropsychological outcome at 4 years.

The persistence and predictive value at 3-5 years of age of three signs detected within the first 18 months of life were investigated: phasic stretch reflex in one or both gastrocnemius muscles, imbalance in passive axial tone with an excess of dorsal extension, and a ridge on the squamous sutures. Phasic stretch reflex and at least one of the other signs were found in 14 children during repeated assessments within the first 18 months. The progress of these children was compared with that of 14 matched controls who had repeatedly normal neurological assessments during the first 18 months in the same clinic in Paris. At the age 3-5 years all the children were then assessed blindly by the second author from a pediatric neurological viewpoint and by two psychologists and two psychomotor therapists as well. The parents of the affected children reported significantly more problems in motor/praxis skills, language development and attention. Abnormal neurological signs were also significantly more frequent than in the controls. Suboptimal cognition did not reach significance. The neurological inclusion criteria were still present at 3-5 years old in 86% (vs. respectively 100% and 93% during the first 18 months of life) of this small group of children, while the cranial suture sign was still present in only 28% (vs. 64%). Findings of these three signs during the first 18 months of life may help in predicting long-term neurobehavioral or long-term neuropsychological problems.