Iron-superoxide dismutase and monodehydroascorbate reductase transcripts accumulate in response to internode rubbing in tomato

A cDNA encoding an iron-superoxide dismutase (Fe-SOD) was isolated by RACE-PCR from a Lycopersicon esculentum cDNA library. The Fe-SOD cDNA consists of a 746-bp open reading frame and is predicted to encode a protein of 249 amino acids with a calculated molecular mass of 27.9 kDa. The deduced amino acid sequence was very similar to other plant Fe-SODs and a potential chloroplastic targeting was found. To study the induction of oxidative burst in response to mechanical stimulation, the accumulation of Fe-SOD and monodehydroascorbate reductase (MDHAR) mRNAs was analysed in response to young growing internode rubbing in tomato plants. Northern analyses show that Fe-SOD mRNA and MDHAR mRNA accumulated in tomato internodes 10 min after the mechanical stimulation. These results suggest that reactive oxygen species are early involved in the response of a plant to a mechanical stimulation, such as rubbing. The nucleotide sequence data reported in this paper will appear in the NCBI Nucleotide Sequence Databases under the accession number AY262025.     

Ion channels formed in planar lipid bilayers by the dipteran-specific Cry4B Bacillus thuringiensis toxin and its alpha1-alpha5 fragment

Trypsin activation of Cry4B, a 130-kDa Bacillus thuringiensis (Bt) protein, produces a 65-kDa toxin active against mosquito larvae. The active toxin is made of two protease resistant-products of ca. 45 kDa and ca. 20 kDa. The cloned 21-kDa fragment consisting of the N-terminal region of the toxin was previously shown to be capable of permeabilizing liposomes. The present study was designed to test the following hypotheses: (1) Cry4B, like several other Bt toxins, is a channel-forming toxin in plannar lipid bilayers; and (2) the 21-kDa N-terminal region, which maps for the first five helices (alpha1-alpha5) of domain 1 in other Cry toxins, and which putatively shares a similar tri-dimensional structure, is sufficient to account for the ion channel activity of the whole toxin. Using circular dichroism spectroscopy and planar lipid bilayers, we showed that the 21-kDa polypeptide existed as an alpha-helical structure and that both Cry4B and its alpha1-alpha5 fragment formed ion channels of 248 +/- 44 pS and 207 +/- 23 pS, respectively. The channels were cation-selective with a potassium-to-chloride permeability ratio of 6.7 for Cry4B and 4.5 for its fragment. However, contrary to the full-length toxin, the alpha1-alpha5 region formed channels at low dose; they tended to remain locked in their open state and displayed flickering activity bouts. Thus, like the full-length toxin, the alpha1-alpha5 region is a functional channel former. A pH-dependent, yet undefined region of the toxin may be involved in regulating the channel properties.     

Modeling the topological organization of cellular processes

The cell as a dynamical system presents the characteristics of having a dynamical structure. That is, the exact phase space of the system cannot be fixed before the evolution and integrative cell models must state the evolution of the structure jointly with the evolution of the cell state. This kind of dynamical systems is very challenging to model and simulate. New programming concepts must be developed to ease their modeling and simulation. In this context, the goal of the MGS project is to develop an experimental programming language dedicated to the simulation of this kind of systems. MGS proposes a unified view on several computational mechanisms (CHAM, Lindenmayer systems, Paun systems, cellular automata) enabling the specification of spatially localized computations on heterogeneous entities. The evolution of a dynamical structure is handled through the concept of transformation which relies on the topological organization of the system components. An example based on the modeling of spatially distributed biochemical networks is used to illustrate how these notions can be used to model the spatial and temporal organization of intracellular processes.     

Characterisation of mutations in 77 patients with X-linked myotubular myopathy,including a family with a very mild phenotype

X-linked myotubular myopathy is characterised by neonatal hypotonia, muscle weakness and respiratory distress in affected males, leading often to early death, although prolonged survival is observed in milder forms, or as a result of prolongation of ventilation support. It is caused by mutations in the MTM1 gene, which encodes a phosphatase called myotubularin, which has been highly conserved during evolution, down to yeasts ( S. cerevisiae and S. pombe). To date, 251 mutations have been identified in unrelated families, corresponding to 158 different disease-associated mutations, which are widespread throughout the gene. We have found additional mutations in 77 patients, including 35 novel ones. We identified a missense mutation N180K in a 67-year-old grandfather (the oldest known patient with an MTM1 mutation), previously suspected to have autosomal centronuclear myopathy, and in his two grandsons also mildly affected. Mild and moderate phenotypes associated with novel missense mutations and with a translation initiation defect mutation are discussed, as well as severe phenotypes associated with particular novel mutations. With the present report, 192 different mutations in the MTM1 gene have been described in 328 families. The spectrum of mutations is now enlarged from the very severe classic neonatal phenotype to very mild phenotype allowing survival to the age of 67 years.     

Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: a structure-stability relationship study and neighbour bases effect

We report herein on the synthesis, the incorporation into triplex forming oligonucleotides (TFO) and the recognition properties of a series of synthetic nucleosides designed for the specific recognition of an inverted A x T base pair in a pyrimidine triple helix motif. These analogues were designed on the basis of the results obtained with our previously reported compounds S and B(t), in order to define a structure-stability relationship. We report also on the chemical nature effect of the bases flanking S in the case of S-containing TFOs, in order to get further informations about the recognition process within the A x TxS triplet. This study establishes guidelines for the conception of more potent analogues for the recognition of both A x T and G x C inverted base pairs.     

New nitrogen mustards structurally related to (L)-carnitine

Enantiopure nitrogen mustards which mimic (L)-carnitine framework are prepared by a multi-step synthesis from the (R)-di-tert-butyl malate and their antitumor properties evaluated.     

HIV-1 entry into T-cells is not dependent on CD4 and CCR5 localization to sphingolipid-enriched,detergent-resistant,raft membrane domains

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56(lck). Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.     

Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.     

Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization

The aim of our study was to determine, as a function of [Cu(2+)]/[LDL] ratios (0.5 and 0.05) and of oxidation phases, the extent of LDL oxidation by assessing the lipid and apo B oxidation products. The main results showed that: (i) kinetics of conjugated diene formation presented four phases for Cu(2+)/LDL ratio of 0.5 and two phases for [Cu(2+)]/[LDL] ratio of 0.05; (ii) oxidation product formation (cholesteryl ester and phosphatidylcholine hydroperoxides, apo B carbonyl groups) occurred early in the presence of endogenous antioxidants, under both copper oxidation conditions; (iii) apo B carbonylated fragments appeared when antioxidants were totally consumed at [Cu(2+)]/[LDL] ratio of 0.5; and (iv) antioxidant concentrations were stable, oxysterol formation was negligible, and no carbonylated fragment was detected at [Cu(2+)]/[LDL] ratio of 0.05. Depending on the copper/LDL ratio, oxidized LDL differ greatly in the nature of lipid peroxidation product and the degree of apo B fragmentation.     

Selective interaction of ethidium derivatives with quadruplexes: an equilibrium dialysis and electrospray ionization mass spectrometry analysis

The telomeric G-rich single-stranded DNA can adopt in vitro an intramolecular quadruplex structure, which has been shown to directly inhibit telomerase activity. The reactivation of this enzyme in immortalized and most cancer cells suggests that telomerase is a relevant target in oncology, and telomerase inhibitors have been proposed as new potential anticancer agents. In this paper, we have analyzed the selectivity of four ethidium derivatives and ethidium itself toward different G-quadruplex species, with electrospray mass spectrometry and competitive equilibrium dialysis and evaluated their inhibitory properties against telomerase. A selectivity profile may be obtained through electrospray ionization mass spectrometry (ESI-MS), which is in fair agreement with competitive equilibrium dialysis data. It also provides unambiguous data on the number of binding sites per nucleic acid (maximal number of two ethidium derivatives per quadruplex, in agreement with external stacking). Our experiments also demonstrate that one compound (4) is the most active and selective G-quadruplex ligand within this series and the most selective telomerase inhibitor in a modified TRAP-G4 assay.