[Evaluation of NT-proBNP as an early biological marker of cardiac dysfunction in septic shock]. / Evaluation du NT-proBNP comme marqueur biologique de la dysfonction cardiaque du choc septique.

OBJECTIVE: To evaluate the NT-proBNP as a biological diagnosis marker of the myocardial dysfunction in septic shock. STUDY DESIGN: Non-randomized prospective clinical study with written assent. The analysis of the data obtained was retrospective. PATIENTS AND METHODS: All the patients with septic shock in the beginning of evolution (less than 24h) were included. Patients with cardiac insufficiency, insufficient respiratory function and chronic renal insufficiency as well as cirrhotic patients were excluded. Among patients in shock, a NT-proBNP concentration measurement and a cardiac echography by transthoracic way were carried out at inclusion. The rates of NT-proBNP were compared with the data of the echography. RESULTS: Thirty-three patients in septic shock were included. On the whole of the collective, whether or not there is a cardiac dysfunction, the rates of NT-proBNP are not significantly different (11,306+/-16,196 pg/ml versus 10,697+/-12,346 pg/ml). By eliminating the patients with severe renal failure, we show that the NT-proBNP is non-significantly increased in the event of right and/or left ventricular failure (5751+/-4180 pg/ml versus 1,256+/-999 pg/ml). CONCLUSION: The NT-proBNP can help to detect the cardiogenic share sometimes implied in the haemodynamic failure of the septic shock. However, because of the influence of the renal insufficiency and the respiratory, cardiologic and hepatic comorbidities on its secretion, its use cannot be recommended for patients in septic shock.

Effect of the polar headgroup of phospholipids on their interaction with actin.

It is generally admitted that actin filaments are anchored to a membrane by membranar actin-binding-proteins. However, we found that actin may also interact directly with membrane phospholipids. The actin-phospholipid complex has been investigated at the air-water interface using a film balance technique. In order to probe the effect of the phospholipid headgroup on the actin-phospholipid interaction, we focus mainly on phospholipids that have the same acyl chain length but different headgroups. For all the phospholipids, the apparent area per molecule (the total surface divided by the number of lipid molecules) increases after the injection of the protein into the subphase, which suggests an intercalation of actin between the phospholipid molecules. This effect seems to be more important for DMPE and DMPS than for DMPG, suggesting that the headgroup plays an important role in this intercalation. The critical surface pressure associated to the liquid expanded-liquid condensed (LE-LC) phospholipid transition increases with the concentration of G-actin and thus suggests that G-actin acts as an impurity, simply competing as a surfactant at the air-water interface. On the other hand, F-actin affects the LE to LC transition of phospholipids differently. In this case, the LE to LC transition is broader and F-actin slightly decreases the critical surface pressure, which suggests that electrostatic interactions are involved.

Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray.

This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue.

Adsorption of actin at the air-water interface: a monolayer study.

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging-plate method. Actin, a very soluble protein, moves from the subphase to the air-water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G-actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F-actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F-actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G-actin film at the beginning of the compression and of an F-actin film at the end of the compression process.

Surface film pressure of actin: interactions with lipids in mixed monolayers.

The interactions of actin with neutral lipid films made from DLPC, and with positively charged films built from DLPC and stearylamine (SA), have been characterized by the monolayer technique. Injection of actin underneath an expanded lipid film produces an increase in the surface pressure that is consistent with a penetration of the lipid molecules by actin. This adsorption of actin to the lipid is more pronounced either with positively charged films or with Mg(2+) present in the sub-phase, suggesting that the mechanism involves an electrostatic attraction. During compression, the actin molecules are squeezed out into the sub-phase, carrying along some lipid molecules; this suggests a strong affinity of the lipids for actin. An analysis of the dilational modulus shows that when actin is found as monomers at the interface, the mixed actin-lipid film undergoes three phase changes upon compression. On the other hand, when actin is polymerized at the interface, the actin and the lipid form a rigid film for which the compressibility is mostly dominated by actin.

Towards the saturation of the pepper linkage map by alignment of three intraspecific maps including known-function genes.

Three populations composed of a total of 215 doubled haploid lines and 151 F2 individuals were used to design an intraspecific consensus map of pepper (Capsicum annuum L.). The individual maps varied from 685 to 1668 cM with 16 to 20 linkage groups (LGs). The alignment of the three individual maps permitted the arrangement of 12 consensus major linkage groups corresponding to the basic chromosome number of pepper and displaying a complex correspondence with the tomato map. The consensus map contained 100 known-function gene markers and 5 loci of agronomic interest (the disease-resistance loci L, pvr2, and Pvr4; the C locus, which determines capsaicin content; and the up locus, controlling the erect habit of the fruits). The locations of three other disease-resistance loci (Tsw, Me3, and Bs3) and the y locus, which determines the yellow fruit colour, were also found on this consensus map thanks to linked markers. Here we report on the first functional detailed map in pepper. The use of candidate gene sequences as genetic markers allowed us to localize four clusters of disease-resistance gene analogues and to establish syntenic relationships with other species.

Unique evolution of neurohypophysial hormones in cartilaginous fishes: possible implications for urea-based osmoregulation.

Most bony vertebrate species display a great evolutionary stability of their two neurohypophysial hormones, so that two molecular lineages, isotocin-mesotocin-oxytocin and vasotocin-vasopressin, have been traced from bony fishes to mammals. Chondrichthyes, in contrast, show a striking diversity of their oxytocin-like hormones, yet show a substantial decrease in vasotocin stored in neurohypophysis when compared to nonmammalian bony vertebrates. In the rays, glumitocin ([Ser(4),Gln(8)]-oxytocin) has been identified. In the spiny dogfish, aspargtocin ([Asn4]-oxytocin) and valitocin ([Val(8)]-oxytocin) have been characterized whereas in the spotted dogfish, asvatocin ([Asn(4),Val(8)]-oxytocin) and phasvatocin ([Phe(3),Asn(4),Val(8)]-oxytocin) have been found. Finally, in the holocephalian Pacific ratfish, oxytocin, the typical peptide of placental mammals, has been discovered. The duplication of the oxytocin-like hormone gene found in dogfishes has been observed only in some Australian and American marsupials. Cartilaginous fishes have developed an original urea-based osmoregulation involving a glutamine-dependent urea synthesis and blood urea retention through renal urea transporters. Furthermore, marine species use a rectal salt gland for sodium chloride excretion. Although vasopressin, in mammals, and vasotocin, in nonmammalian tetrapods, are clearly implied in water and salt homeostasis, the hormones involved in the blood osmotic pressure regulation of elasmobranchs are still largely unknown. It is suggested that the great diversity of oxytocin-like hormones in elasmobranchs expresses a release from an evolutionary receptor-binding constraint, so that amino-acid substitutions reflect neutral evolution. In contrast, the preservation of vasotocin suggests a selective pressure, which may be related to the regulation of renal urea transporter-recruitment mechanisms, as it has been shown for vasopressin in mammals. J. Exp. Zool. 284:475-484, 1999.

Interaction between G-actin and various types of liposomes: A 19F, 31P, and 2H nuclear magnetic resonance study.

We have investigated in the present study the interaction between G-actin and various types of liposomes, zwitterionic, positively charged, and negatively charged. To investigate at the molecular level the conformation of actin in the presence of lipids, we have selectively attached a fluorinated probe, 3-bromo-1,1,1-trifluoropropanone, to the actin cysteine residues 10, 285, and 374 and used high-resolution 19F nuclear magnetic resonance spectroscopy to investigate the probe resonances. The results indicate a change in the mobility of the 19F labels when G-actin is in the presence of positively charged liposomes made of DMPC and stearylamine and in the presence of DMPG, a negatively charged lipid. No conformational change was observed in the actin molecule in the presence of neutral liposomes. Electron micrographs of these systems reveal the formation of paracrystalline arrays of actin filaments at the surface of the positively charged liposomes, while no evidence of actin polymerization or paracrystallization was observed in the presence of DMPG. The interaction between actin and the lipid polar headgroup has also been investigated using solid-state phosphorus and deuterium NMR. The results indicate no evidence of interaction between actin and zwitterionic liposomes but show an interaction between the positively charged liposomes and a negative charge on the actin molecules. Interestingly, the negatively charged liposomes interact with a positive charge, which is most likely associated with the three residues (His-Arg-Lys) preceding the cysteine 374 residue in the protein.

Adaptive evolution of water homeostasis regulation in amphibians: vasotocin and hydrins.

Volemia and osmolality homeostasis is ensured in vertebrates through neuroendocrine reflexes, involving an afferent neural branch from baro- and osmo-receptors to hypothalamus and an efferent endocrine branch from secretory neurons to target hydroosmotic cells equipped with receptors and effectors. Whereas the osmoregulatory system in the tadpole comprises three organs, namely gut, kidney and gills, as in freshwater fishes, the adult displays a quaternary strategy with gut, kidney, urinary bladder and skin. In particular, the cutaneous permeability entails a great evaporative water loss when the animal is in the open air, loss that must be compensated by water reabsorption through the nephron and the urinary bladder and mainly by water uptake through the skin. Adaptation occurred at the level of these organs by regulation of their permeability through neurohypophysial hormones. Aside from vasotocin, active on the three organs, all anuran Amphibia possess hydrin 2 (vasotocinyl-Gly), a peptide resulting from a down-regulation of provasotocin processing. Exceptionally Xenopus laevis, a permanent aquatic toad, has hydrin 1 (vasotocinyl-Gly-Lys-Arg) instead of hydrin 2. Hydrins are somewhat more active than vasotocin on water permeation of skin and bladder but are devoid of antidiuretic activity. Adaptive evolution has created, along with the vasotocin-nephron system, preserved in all terrestrial non-mammalian tetrapods, additional functions such as the hydrin-skin and hydrin-bladder rehydration mechanisms. Specific hydrin receptors might exist in the skin and the bladder, different from those of vasotocin in the kidney. It is assumed that the water channel recruitment mechanism, found for vasopressin acting on the collecting duct principal cells in mammals, is also involved when vasotocin and hydrins stimulate their hydroosmotic target cells and that hormone-regulated aquaporin 2-like proteins could be identified in the three osmoregulatory organs of amphibians.

Distinct hydro-osmotic receptors for the neurohypophysial peptides vasotocin and hydrins in the frog Rana esculenta.

The biological properties of vasotocin, hydrin 1 (vasotocinyl-Gly-Lys-Arg) and hydrin 2 (vasotocinyl-Gly), in particular the hydro-osmotic activities on the frog skin, the frog urinary bladder and the frog kidney, have been compared. Hydrins are as active or more active than vasotocin on the first two organs but they are virtually devoid of antidiuretic activity in the rat and the frog, in contrast to vasotocin. It appears that where the oxytocin ring (residues 1-6), present in the three peptides, is necessary for the action on the three organs, the C-terminal amidated group of vasotocin is necessary for the renal receptor but not for the skin and bladder receptors. It is known that amphibians have two types of vasotocin receptors, V1 and V2, homologous to the vascular/hepatic V1 and the renal V2 vasopressin receptors of mammals, respectively. We suggest that adaptation has led to specialization of (at least) two subtypes of hydro-osmotic V2 receptors, the renal subtype on which vasotocin is mainly active for the reabsorption of tubular water, and the skin/bladder subtype on which hydrin 2 is specifically involved in ensuring the rehydration of the animal. Cooperative evolution might have created in anuran Amphibia, on the one hand, two hydro-osmotic peptides, vasotocin and hydrin 2, derived from a single precursor through differential processing; and on the other hand, two corresponding receptors in kidney and skin for internal and external water recovery.

The neurohypophysial endocrine regulatory cascade: precursors, mediators, receptors, and effectors.

The neurohypophysial endocrine regulatory cascade has been described as a molecular model of neuroendocrine control of organismal functions. Any physiological function can be analyzed in molecular terms as a succession of interactions occurring either in a solution or in a membrane system. The key mechanism in the ordering of the cascade is the conformational recognition of the two partners at each step. Each interaction results in a change of conformation of a recognized protein that in turn becomes a recognizer for the following molecule. The cascade starts within the secretory cell by the processing of the expressed precursor along the secretory pathway until the storage of the mature mediator in vesicles and its subsequent exocytic secretion in blood. The circulating mediator recognizes the target cell through specific membrane receptors that transduce the message within this target cell. A second intracellular cascade leads to activation of the effector, the protein fulfilling the physiological function. The complexity of the messages is, in part, due to the duplication propensity of the genomic DNA, the frequent occurrence of multiple copies for precursors, mediators, receptors, and effectors, and therefore, a combinatorial diversity that increases during the course of evolution. Vertebrate neurohypophysial hormones can be ordered in two main evolutionary lineages, culminating in oxytocin and vasopressin in placental mammals. In this field, diversification of the messages was made by differential processing of the precursors, secondary gene duplications, the emergence of several types of receptors for each hormone, and a variety of effectors triggered by the second messengers within differentiated target cells. This review is an attempt to integrate neurohypophysial functions at the molecular, cellular, and organismal levels.

A new neurohypophysial peptide, seritocin ([Ser5,Ile8]-oxytocin), identified in a dryness-resistant African toad, Bufo regularis.

From the pituitary neurointermediate lobe of the African toad Bufo regularis, vasotocin, hydrin 2 (vasotocinyl-Gly) and a mesotocin-like peptide have been isolated by HPLC and characterized by mass spectrometry, amino acid sequence and chromatographic coelution with synthetic peptides. The mesotocin-like peptide has been identified as [Ser5,Ile8]-oxytocin in place of mesotocin ([Ile8]-oxytocin) found in all other amphibians investigated to date. The name seritocin is suggested. The molecule is virtually devoid of oxytocic activity on rat uterus in contrast to mesotocin. On the other hand, the molar ratio of hydrin 2 to vasotocin in the pituitary reaches 2, whereas it is about 1 in toads and frogs from temperate regions. B. regularis is an anuran species able to withstand a hot and dry season by burrowing. The possible relationship between occurrence of seritocin and adaptation to arid environment remains to be demonstrated.

Man and the chimaera. Selective versus neutral oxytocin evolution.

The oxytocin/vasopressin superfamily encompasses vertebrate and invertebrate peptides and therefore the ancestral gene encoding the precursor protein antedates the divergence between the two groups, about 700 million years ago. The preserved nonapeptide pattern indicates that both the precursor structures and the processing enzymatic machinery were greatly conserved to ensure the building of a specific conformation. Substitutions, which may be neutral or selective, occurred in precise positions. Virtually all vertebrate species possess an oxytocin-like and a vasopressin-like peptide so that two evolutionary lineages can be traced. Because a single peptide, vasotocin ([Ile3]-vasopressin or [Arg8]-oxytocin) has been found in the most primitive Cyclostomata, a primordial gene duplication and subsequent mutations are assumed to have given rise to the two lineages. They started with vasotocin and isotocin ([Ser4,Ile8]-oxytocin) in bony fishes and culminated with vasopressin and oxytocin in placental mammals. Mesotocin ([Ile3]-oxytocin), found in lungfishes, amphibians, reptiles, birds and marsupials, appears as an evolutionary intermediate. The change from isotocin ([Ser4,Ile8]-oxytocin) into mesotocin ([Ile8]-oxytocin), can be observed in African and Australian lungfishes, species making the transition from bony fishes to land vertebrates. On the other hand the replacement of mesotocin by oxytocin can be detected in marsupials, particularly in the North-American opossum and the Australian Northern bandicoot that have both mesotocin and oxytocin whereas placental mammals possess only oxytocin. The invariability of this peptide in placentals can be explained by receptor-fitting selective pressure. In contrast to bony vertebrates in which neurohypophysial hormones revealed a remarkable structural stability, cartilaginous fishes displayed an unique oxytocin-like hormone evolution with variability and duality. Aside from vasotocin, in the subclass Selachii, rays have glumitocin ([Gln8-oxytocin]) and sharks possess two peptides: aspargtocin ([Asn4-oxytocin]) and valitocin ([Val8-oxytocin]) for the spiny dogfish, asvatocin ([Asn4,Val8]-oxytocin) and phasvatocin ([Phe3,Asn4,Val8]-oxytocin) for the spotted dogfish. In the other subclass Holocephali, the chimaera (ratfish) has oxytocin, the typical hormone of placental mammals. Cartilaginous fishes used urea rather than salts for their osmoregulation and oxytocin-like hormones could have been relieved from osmoregulatory functions and able to accept many neutral variations.

Special evolution of neurohypophysial hormones in cartilaginous fishes: asvatocin and phasvatocin, two oxytocin-like peptides isolated from the spotted dogfish (Scyliorhinus caniculus).

In contrast to most vertebrate species that possess one oxytocin-like hormone and one vasopressin-like hormone, a few groups, such as marsupials or cartilaginous fishes, are endowed with two peptides of either or both types, suggesting possible gene duplications. We have now isolated two oxytocin-like hormones from the pituitary of the spotted dogfish Scyliorhinus caniculus (suborder Galeoidei). Microsequencing as well as chromatographic and pharmacological comparisons with synthetic peptides show that these peptides are [Asn4,Val8]oxytocin (asvatocin) and [Phe3,Asn4,Val8]-oxytocin (phasvatocin). Asvatocin and phasvatocin display oxytocic activity on rat uterus, about 80 and 5 milliunits per nmol, respectively, and virtually no pressor activity on anesthetized rats. They occur in roughly equal molar amounts in the gland; vasotocin is also present in a proportional amount that is lower by about a factor of 20. In addition to the duality, conservative amino acid substitutions are observed in the two oxytocic peptides in positions 4 (Gln-4-->Asn) and 8 (Leu-8-->Val), when compared with oxytocin. Furthermore, replacement of the isoleucine residue found in position 3 of all other oxytocin-like hormones by phenylalanine in phasvatocin is exceptional; it determines a dramatic decrease of the oxytocic activity. Preservation of the C-terminal-amidated nonapeptide pattern in the 12 vertebrate neurohypophysial hormones known to date suggests that both precursors and processing enzymes have coevolved tightly. On the other hand, whereas the great evolutionary stability of the mature hormones (generally observed in vertebrates) suggests a strict messenger-receptor coevolution, the exceptional diversity found in cartilaginous fishes (six oxytocin-like peptides identified out of eight known) might be due to a looseness of selective constraints, perhaps in relationship with their specific urea osmoregulation.

Quantification of superantigen induced IFN-gamma production by computerised image analysis--inhibition of cytokine production and blast transformation by pooled human IgG.

A quantitative image analysis technique was developed to assess the cytokine content of immunocytochemically stained cytokine producing cells. Peripheral blood mononuclear cells were stimulated to induce cytokine production with the superantigen streptococcal pyrogenic exotoxin-A. We have developed a method based on indirect immunocytochemistry which identifies IFN-gamma producing cells by a characteristic morphology generated by the accumulation of IFN-gamma in the Golgi organelle. An image analysis technique permitted discrimination between these producer cells and IFN-gamma binding target cells, which showed a different appearance, with staining restricted to the cell surface membrane. A semi-automated routine programme allowed the signal from a video camera to be processed by computerised image analysis methodology. This enabled us to measure the number of cytokine producing cells, the cytokine staining intensity in individual cells and the cell size expressed in actual cell area. The incidence of IFN-gamma producing cells determined by image analysis measurement was compared to results obtained using manual microscopy. Cell size was assessed by the image analysis system as well as by flow cytometry. Administration of pooled human IgG for intravenous use (IVIg) to the superantigen stimulated cells significantly down-regulated IFN-gamma production, both in terms of the numbers of producer cells and in terms of cytokine staining intensity in individual cells. In addition blast transformation of cells was substantially reduced. These effects, mediated by IVIg, were also evident following delayed IVIg administration 24 h after the initial cell stimulation.

Action of neurohypophysial granule Lys-Arg endopeptidase on synthetic polypeptides comprising the processing sequence of provasopressin-neurophysin.

Neurohypophysial granule Ca(2+)-dependent endopeptidases have been allowed to act on synthetic polypeptides derived from the N-terminal sequence of bovine provasopressin-neurophysin, namely vasopressinyl-glycyl-lysyl-arginyl-alanylamide and vasopressinyl-glycyl-lysyl-arginyl-alanyl-methionyl-serinamide+ ++. Membrane-bound enzymes have been used at pH 5.5 for 16 hr at 37 degrees C. Products have been identified by high-pressure liquid chromatography (HPLC) and by mass spectrometry performed on substances isolated by HPLC. With both substrates, vasopressinyl-Gly-Lys-Arg(OH) has been identified as a product confirming the Lys-Arg specificity previously observed on small peptide fluorogenic substrates. Cleavage yields, however, appear low suggesting that some factors are missing, for example a targeting action of the precursor neurophysin domain to the granule membrane.

Bony fish neurophysins. Identification of MSEL- and VLDV-neurophysins of the pollack (Pollachius virens).

The two types of neurophysins known in vertebrate species, namely MSEL-neurophysin (vasopressin-like hormone-associated neurophysin) and VLDV-neurophysin (oxytocin-like hormone-associated neurophysin) have been purified from the pollack (Pollachius virens) pituitary through a combination of molecular sieving and high-pressure liquid chromatography (HPLC). Homogeneity has been checked by gel electrophoresis and return in HPLC. The apparent molecular masses measured by SDS-electrophoresis are near 12 kDa, significantly higher than those found for their mammalian homologues (10 kDa). The two types of neurophysins have been recognized through their N-terminal amino acid sequences. The primary structure of MSEL-neurophysin has been partially determined using automated Edman degradation applied on native and reduced-alkylated protein, as well as peptides derived by trypsin or staphylococcal proteinase hydrolyses. Comparison of pollack MSEL-neurophysin with ox, goose and frog counterparts reveals that particular positions in the polypeptide chain are subjected to substitutions and that the numbers of substitutions do not seem closely related to the paleontological times of divergence between the different vertebrate classes.

Chemical identification of the mammalian oxytocin in a holocephalian fish, the ratfish (Hydrolagus colliei).

The neurohypophysial hormones of the ratfish (Hydrolagus colliei), a species belonging to the subclass Holocephali of cartilaginous fishes, have been investigated. An oxytocin-like hormone has been isolated from acetone-desiccated pituitary glands by using successively molecular sieving and high-pressure liquid chromatography. The peptide has been identified as oxytocin by coelution with synthetic oxytocin in HPLC, amino acid sequencing, mass spectrometry, and C-terminal sequencing through carboxypeptidase Y. Vasotocin may be present in a very small amount. Cartilaginous fishes appear to display a great diversity in their oxytocin-like hormones since five different peptides have been identified in rays and sharks that belong to the second subclass Selachii.