Neurosteroid modulation of the presynaptic NMDA receptors regulating hippocampal noradrenaline release in normal rats and those exposed prenatally to diazepam

Prenatal exposure to diazepam (DZ), a positive allosteric modulator of the gamma-aminobutyric acid(A) (GABA(A)) receptor complex, exerts profound effects that become more evident during puberty and in many cases are sex-specific, suggesting that such exposure interferes with the activity of steroid hormones. Apart from their well known effects on the genome, the reduced metabolites of many steroid hormones also interact directly with membrane receptors, including those for N-methyl-D-aspartate (NMDA). In this study, we compared the effects of several neurosteroids on NMDA receptors from normal rats and those exposed in utero to DZ (1.25mg/kg per day) from the 14th through the 20th day of gestation.In superfused rat hippocampal synaptosomes, activation of the NMDA receptor stimulates the basal release of [3H]noradrenaline ([3H]NA), which was used in our study as an index of receptor function. [3H]NA release was evoked in a concentration-dependent manner by NMDA (100 microM) plus glycine (GLY). The maximal increase (68.23+/-3.86%) with respect to basal release was achieved with a GLY concentration of 10 microM, and the EC(50) for GLY was 0.1 microM. Release stimulated by 100 microM NMDA + 0.1 microM GLY was not modified by any of the neurosteroids tested, with the exception of pregnenolone sulfate (PREG-S), which produced a 78.57+/-3.94% reduction in release at the maximal concentration used (0.3 microM). In synaptosomes from animals exposed in utero to DZ, the inhibitory effect of PREG-S was reduced by 46.55+/-2.33%.Given the important roles played by NMDA receptors in physiological and pathological processes within the central nervous system (CNS), characterization of NMDA receptor modulation is an important objective. The fact that this modulation can be altered by exposure in utero to DZ indicates that the behavioral abnormalities observed in exposed animals might be partially attributed to an altered sensitivity of NMDA receptors to the modulatory effects of neurosteroids.     

Demonstration of in vitro interaction between tumor suppressor lysyl oxidase and histones H1 and H2: definition of the regions involved

Lysyl oxidase (LOX) is the enzyme that cross-links extracellular collagen and tropoelastin and is involved in tumor suppressor activity. Based on the existent homologies between lysine-rich regions of tropoelastin and the "lysine-rich" histone H1, we tested the possibility that H1 could be a new nuclear target. Our study shows that LOX could actually interact specifically not only with histone H1, but also with histone H2. Mechanisms and significance of these interactions are discussed in detail.     

Atherina punctata and Atherina lagunae (Pisces,Atherinidae), new species found in the Mediterranean sea. 2. Molecular investigations of three Atherinid species

On the basis of morphoanatomical parameters, the sand smelt species (Atherina boyeri Risso, 1810) is viewed as a highly polymorphic complex. In this study, intraspecific sequence variation in a portion of the cytochrome b gene was examined in 88 individuals from Tunisia and France. The correlation between the results of statistical analysis of the sequence data using a variety of tree-building algorithms and morphoanatomical analyses demonstrated the subdivision into three putative species: A. boyeri, which only includes non-punctuated fishes, A. punctata, which corresponds to punctuated fishes and A. lagunae, which corresponds to atherines living in lagoons.     

alpha1-Adrenoceptor antagonists. 5. Pyridazinone-arylpiperazines. Probing the influence on affinity and selectivity of both ortho-alkoxy groups at the arylpiperazine moiety and cyclic substituents at the pyridazinone nucleus

Our previous work on pyridazinone-arylpiperazine derivatives suggested some structural features that a compound should have to show high affinity and good selectivity for alpha(1) adrenoceptors (AR) with respect to alpha(2)-AR. Accordingly, two classes of new alkoxyphenylpiperazinylheptylpyridazinones were designed and synthesized to evaluate the effect of the alkoxy substituent on affinity and selectivity. As expected, affinity increased with larger alkoxy groups. Affinity values are all comparable with that of the reference compound (prazosin), with the exception of compound 1c found 4.5-fold more active than prazosin.     

Discovery and analysis of antisense oligonucleotide activity in cell culture

In the past decade antisense oligonucleotides (ASOs) have proven to be a useful tool for dissection of gene function in molecular cell biology (Koller, E., Gaarde, W. A., and Monia, B. P. (2000) Trends Pharm. Sci., 21, 142-148), and validation of gene targets in animal models (Crooke, S. T. (1998) Biotechnol. Gen. Eng. Rev. 15, 121-157), as well as a means for therapeutic treatment of human diseases (Bennett, C. F. (1999) Exp. Opin. Invest. Drugs 8, 237-253). An important step toward usage of ASOs in the described applications is identification of an active ASO. This article describes the underlying basis and means for achieving this goal in cell culture.     

Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast antioxidant functional in pathogen defense

Peroxiredoxins are ubiquitous thioredoxin- or glutaredoxin-dependent peroxidases, the function of which is to destroy peroxides. Peroxiredoxin Q, one of the four plant subtypes, is a homolog of the bacterial bacterioferritin comigratory proteins. We show here that the poplar (Populus tremula x Populus tremuloides) protein acts as a monomer with an intramolecular disulfide bridge between two conserved cysteines. A wide range of electron donors and substrates was tested. Unlike type II peroxiredoxin, peroxiredoxin Q cannot use the glutaredoxin or cyclophilin isoforms tested, but various cytosolic, chloroplastic, and mitochondrial thioredoxins are efficient electron donors with no marked specificities. The redox midpoint potential of the peroxiredoxin Q catalytic disulfide is -325 mV at pH 7.0, explaining why the wild-type protein is reduced by thioredoxin but not by glutaredoxin. Additional evidence that thioredoxin serves as a donor comes from the formation of heterodimers between peroxiredoxin Q and monocysteinic mutants of spinach (Spinacia oleracea) thioredoxin m. Peroxiredoxin Q can reduce various alkyl hydroperoxides, but with a better efficiency for cumene hydroperoxide than hydrogen peroxide and tertiary butyl hydroperoxide. The use of immunolocalization and of a green fluorescence protein fusion construct indicates that the transit sequence efficiently targets peroxiredoxin Q to the chloroplasts and especially to those of the guard cells. The expression of this protein and of type II peroxiredoxin is modified in response to an infection by two races of Melampsora larici-populina, the causative agent of the poplar rust. In the case of an hypersensitive response, the peroxiredoxin expression increased, whereas it decreased during a compatible interaction.     

Glucose isomerase of the Streptomyces sp. SK strain: purification, sequence analysis and implication of alanine 103 residue in the enzyme thermostability and acidotolerance

The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.     

Characterization of ferredoxin:thioredoxin reductase modified by site-directed mutagenesis

Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.     

Phaeodactylum tricornutum: An established model species for diatom molecular research and an emerging chassis for algal synthetic biology

Diatoms are prominent and highly diverse microalgae in aquatic environments. Compared with other diatom species, Phaeodactylum tricornutum is an “atypical diatom” displaying three different morphotypes and lacking the usual silica shell. Despite being of limited ecological relevance, its ease of growth in the laboratory and well-known physiology, alongside the steady increase in genome-enabled information coupled with effective tools for manipulating gene expression, have meant it has gained increased recognition as a powerful experimental model for molecular research on diatoms. We here present a brief overview of how over the last 25 years P. tricornutum has contributed to the unveiling of fundamental aspects of diatom biology, while also emerging as a new tool for algal process engineering and synthetic biology.