Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

The agrochemical arsenal versus the enemies of plants. General considerations

Plants are attacked, not only by various microorganisms, but also by other enemies, such as molluscs, nematods, mites, and insects. They have evolved complex and efficient mechanisms to defend themselves against pathogens (hypersensitive response, systemic acquired resistance) and herbivores (release of volatile compounds that attract predators of the herbivores, accumulation of proteinase inhibitors). Yet, the confrontation of the plants with their invaders can also turn to the advantage of the latter. In the past, the attacks of crops regularly brought about dramatic economic losses. From the World War II onwards, the development of organic chemistry associated with a growing awareness of the problems of agriculture has resulted in the production of a constantly growing number of plant protection products. They are currently divided into about ten classes, the herbicides, fungicides, and insecticides-acaricides making up more than 90% of the world market. Most of the agrochemical products put on the market over these last three decades are used in relatively low doses and have a more favourable toxicological and ecotoxicological profile than those of the former pesticides, many of which are now withdrawn from the market. Several more or less recent families are derivatives of metabolites from various organisms. Thus, the improvement achieved in the protection of crops is outstanding. However, one on the main side-effect is an environmental imbalance that has entailed a dependency on agrochemicals. Quite judiciously, alternative strategies (elicitors, genetic engineering, etc.) have been initiated or developed over the last decade.     

Sustainable management of fixed dunes: example of a pilot site in Brittany (France)

The sand-dunes of Quiberon was chosen as a pilot site to investigate experimentation in conservatory management. Sand burial is necessary to conserve the semi-fixed dune which is a transitory dynamic stage. In the fixed dune, low disturbances benefit the vegetation diversity while heavy ones create serious injury. An opening of the milieu can restore very fast but a naked substrate is difficult to heal. The deposition of gorse branches is then efficient to facilitate the restoration. The fixed dune biodiversity is linked to human activities. Disturbances, natural or not, may be used as management tools.     

Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing

Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2'-amino-3'-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H(2)O(2) resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H(2)O(2) induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEK(CA)), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells, whereas it quickly colocalized with actin and stress fibers upon stimulation of ERK by H(2)O(2) or by expression of MEK(CA). We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.     

Analyzing somaclonal variation in micropropagated bananas (<Emphasis Type="Italic">Musa</Emphasis> spp.)

Summary In a micropropagation program, where it is of paramount importance to produce true-to-type planting material, somaclonal variation of any kind is undesirable. Variation among plants regenerated from tissue culture is termed ‘somaclonal variation’. In banana, somaclonal variants of different type have been reported with regard to plant morphology. This article discusses various factors due to which somaclonal variations may arise. Somaclonal variation may be detected by visual screening or by using molecular markers such as randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and by cytological studies. Although somaclonal variation is undesirable in the context of micropropagation, it can be used to advantage for genetic improvement of banana, as has been described.     

Haemoglobin biosynthesis site in rabbit embryo erythroid cells

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.     

Synthesis,structure-activity relationship and in vitro evaluation of coelenterazine and coelenteramine derivatives as inhibitors of lipid peroxidation

Coelenterazine (2-p -hydroxybenzyl-6-(3'-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazolo[1,2-a]pyrazin-3-one, CLZn) and coelenteramine (2-amino-3-benzyl-5-(4'-hydroxyphenyl)-1,4-pyrazine CLM), first described as luciferin and etioluciferin, respectively, of bioluminescent systems in marine organisms are endowed with antioxidant properties. This study was aimed at understanding the structural basis of their chain-breaking properties and at designing new compounds with improved antioxidative properties. For this, a series of 2-amino-1,4-pyrazine derivatives and their related imidazolopyrazinones were synthesised and examined for their capacity to inhibit lipid peroxidation in linoleate micelles subjected to the peroxidizing action of AAPH. Structure-activity relationship studies indicated that the reduction of the peroxidation rate by CLM is mainly determined by the concomitant presence of 5-p-hydroxyphenyl and 2-amino groups in para position. The lipophilic character of substituents also affected this effect. All imidazolopyrazinones induced a lag-time before the onset of the peroxidation process. The hetero-bicyclic imidazolopyrazinone moiety appears as the main contributor to this activity while phenol groups play little role in it. On the other hand, phenol groups were required for the reduction of the peroxidation rate after the lag-phase. The introduction of a supplementary p-hydroxyphenyl substituent at C₈ position did not increase chain-breaking properties. The substitution of the C₅-p-hydroxyphenyl with a catechol moiety or the introduction of a second amino group on the pyrazine ring yielded the most active compounds, superior to imidazolopyrazinones and reference antioxidants like epigallocatechin gallate, vitamin E and trolox. The strong antioxidant properties of 2,6-diaminopyrazines are not dependent on the presence of hydroxyl groups indicating that their reaction mechanism differs from that of 2-amino-1,4-pyrazine derivatives.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.