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.     

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.     

The phage T4 restriction endoribonuclease RegB: a cyclizing enzyme that requires two histidines to be fully active

The regB gene, from the bacteriophage T4, codes for an endoribonuclease that controls the expression of a number of phage early genes. The RegB protein cleaves its mRNA substrates with an almost absolute specificity in the middle of the tertranucleotide GGAG, making it a unique well-defined restriction endoribonuclease. This striking protein has no homology to any known RNase and its catalytic mechanism has never been investigated. Here, we show, using ³¹P nuclear magnetic resonance (NMR), that RegB produces a cyclic 2′,3′-phosphodiester product. In order to determine the residues crucial for its activity, we prepared all the histidine-to- alanine point mutants of RegB. The activity of these mutants was characterized both in vivo and in vitro. In addition, their binding capability was quantified by surface plasmon resonance and their structural integrity was probed by ¹H/¹⁵N NMR correlation spectroscopy. The results obtained show that only the H48A and the H68A substitutions significantly reduce RegB activity without changing its ability to bind the substrate or affecting its overall structure. Altogether, our results define RegB as a new cyclizing RNase and present His48 and His68 as potent catalytic residues. The effect of the in vivo selected R52L mutation is also described and discussed.     

Three dimensional modeling of the cerebrospinal fluid dynamics and brain interactions in the aqueduct of sylvius

A computational fluid dynamics (CFD) method is presented to investigate the flow of cerebro-spinal fluid (CSF) in the cerebral aqueduct. In addition to former approaches exhibiting a rigid geometry, we propose a model which includes a deformable membrane as the wall of this flow channel. An anatomical shape of the aqueduct was computed from magnetic resonance images (MRI) and the resulting meshing was immersed in a marker-and-cell (MAC) staggered grid for to take into account fluid-structure interactions. The time derivatives were digitized using the Crank-Nicolson scheme. The equation of continuity was modified by introducing an artificial compressibility and digitized by a finite difference scheme. Calculations were validated with the simulation of laminar flow in a rigid tube. Then, comparisons were made between simulations of a rigid aqueduct and a deformable one. We found that the deformability of the walls has a strong influence on the pressure drop for a given flow.     

The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies

The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C(alpha) positions is 0.39A). Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.     

Effect of hormones and growth factors on the proliferation of adult cricket neural progenitor cells in vitro

In the adult cricket brain, a cluster of neuroblasts produces new interneurons that integrate into the mushroom body (MB), the main associative structure for multisensory information of the insect brain. In previous study we showed the antagonist role of the two morphogenetic hormones, juvenile hormone (JH) and ecdysone, on the regulation of adult MB neurogenesis in vivo. In order to examine whether these hormones act directly on neural progenitor cells, we developed an organotypic culture of MB cortices. Cell proliferation was assessed by 5-bromo, 2'-deoxyuridine (BrdU) incorporation. We showed that JH increased mushroom body neuroblast (MBNb) proliferation, confirming the mitogenic effect of JH observed in vivo. By contrast, ecdysone did not affect the amount of BrdU-labeled nuclei, suggesting that the inhibitory effect observed in vivo probably proceeded from an indirect pathway. We then examined the role of growth factors known to stimulate neural stem cell/progenitor cell proliferation in vertebrates. As shown by calcium imaging, MBNb only expressed functional receptors for insulin whereas mature interneurons responded to IGF-I and bFGF. Both insulin (10 microg/ml) and IGF-I (10 ng/ml) enhanced MB progenitor cell proliferation in culture, although the insulin effect was more pronounced. This effect was abolished when an inhibitor of polyamine biosynthesis was present in the medium, suggesting a link between polyamines and the insulin signaling pathway. By contrast, bFGF (20-200 ng/ml) failed to stimulate MBNb proliferation. Our results point to conserved and divergent mechanisms between vertebrates and invertebrates in the regulation of adult neural progenitor cell proliferation.     

DeltaBAFF,an alternate splice isoform that regulates receptor binding and biopresentation of the B cell survival cytokine,BAFF

The tumor necrosis family member BAFF is limiting for the survival of follicular B lymphocytes, but excessive BAFF signaling can lead to autoimmunity, suggesting that its activity must be tightly regulated. We have identified a conserved alternate splice isoform of BAFF, called deltaBAFF, which lacks 57 nt encoding the A-A1 loop and is co-expressed with BAFF in many mouse and human myeloid cells. Mouse deltaBAFF appears on the plasma membrane, but unlike BAFF it is inefficiently released by proteolysis. DeltaBAFF can associate with BAFF in heteromultimers and diminish BAFF bioactivity and release. Thus, alternative splicing of the BAFF gene suppresses BAFF B cell stimulatory function in several ways, and deltaBAFF may promote other functions as well.     

Identification of the binding sites of the SR49059 nonpeptide antagonist into the V1a vasopressin receptor using sulfydryl-reactive ligands and cysteine mutants as chemical sensors

To identify the binding site of the human V1a vasopressin receptor for the selective nonpeptide antagonist SR49059, we have developed a site-directed irreversible labeling strategy that combines mutagenesis of the receptor and use of sulfydryl-reactive ligands. Based on a three-dimensional model of the antagonist docked into the receptor, hypothetical ligand-receptor interactions were investigated by replacing the residues potentially involved in the binding of the antagonist into cysteines and designing analogues of SR49059 derivatized with isothiocyanate or alpha-chloroacetamide moieties. The F225C, F308C, and K128C mutants of the V1a receptor were expressed in COS-7 or Chinese hamster ovary cells, and their pharmacological properties toward SR49059 and its sulfydryl-reactive analogues were analyzed. We demonstrated that treatment of the F225C mutant with the isothiocyanate-derivative compound led to dose-dependent inhibition of the residual binding of the radio-labeled antagonist [125I]HO-LVA. This inhibition is probably the consequence of a covalent irreversible chemical modification, which is only possible when close contacts and optimal orientations exist between reactive groups created both on the ligand and the receptor. This result validated the three-dimensional model hypothesis. Thus, we propose that residue Phe225, located in transmembrane domain V, directly participates in the binding of the V1a-selective nonpeptide antagonist SR49059. This conclusion is in complete agreement with all our previous data on the definition of the agonist/antagonist binding to members of the oxytocin/vasopressin receptor family.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.