Valine 10 may act as a driver for product release from the active site of human glutathione transferase P1-1

We have probed the electrophilic binding site (H-site) of human glutathione transferase P1-1 through mutagenesis of two valines, Val 10 and Val 35, into glycine and alanine, respectively. These two residues were previously shown to be the only conformationally variable residues in the H-site and hence may play important roles in cosubstrate recognition and/or product dissociation. Both of these mutant enzymes have been expressed in Escherichia coli and purified and their kinetic properties characterized. The results demonstrate that Val35Ala behaves similarly to wild-type, whereas Val10Gly exhibits a strong decrease of k(cat) and k(cat)/K(m) (cosub) toward two selected cosubstrates: ethacrynic acid and 1-chloro-2,4-dinitrobenzene. Pre-steady-state kinetic analysis of the GSH conjugation with ethacrynic acid shows that both wild-type and Val10Gly mutant enzymes exhibit the same rate-limiting step: the dissociation of product. However, in the Val10Gly mutant there is an increased energetic barrier which renders the dissociation of product more difficult. Similar results are found for the Val10Gly mutant with 1-chloro-2,4-dinitrobenzene as cosubstrate. With this latter cosubstrate, Val 10 also exerts a positive role in the conformational transitions of the ternary complex before the chemical event. Crystallographic analysis of the Val10Gly mutant in complex with the inhibitor S-hexyl-GSH suggests that Val 10 optimally orientates products, thus promoting their exit from the active site.     

A high-resolution linkage map of the citrus tristeza virus resistance gene region in Poncirus trifoliata (L.) Raf.

Resistance to citrus tristeza virus (CTV) was evaluated in 554 progeny of 10 populations derived from Poncirus trifoliata. A dominant gene (Ctv) controlled CTV resistance in P. trifoliata. Twenty-one dominant PCR-based DNA markers were identified as linked to Ctv by bulked segregant analysis. Of the 11 closest markers to Ctv, only 2 segregated in all populations. Ten of these markers were cloned and sequenced, and codominant RFLP markers were developed. Seven RFLP markers were then evaluated in 10 populations. Marker orders were consistent in all linkage maps based on data of single populations or on combined data of populations with similar segregation patterns. In a consensus map, the six closest marker loci spanned 5.3 cM of the Ctv region. Z16 cosegregated with Ctv. C19 and AD08 flanked Ctv at distances of 0.5 and 0.8 cM, respectively. These 3 markers were present as single copies in the Poncirus genome, and could be used directly for bacterial artificial chromosome library screening to initiate a walk toward Ctv. BLAST searches of the GenBank database revealed high sequence similarities between 2 markers and known plant disease resistance genes, indicating that a resistance gene cluster exists in the Ctv region in P. trifoliata.     

The influence of mandibular implant-retained overdentures in masticatory efficiency

doi: 10.1111/j.1741‐2358.2011.00539.x The influence of mandibular implant‐retained overdentures in masticatory efficiency Objective: To evaluate the masticatory efficiency of patients rehabilitated with conventional dentures (CDs) or implant‐retained mandibular overdentures. Background: Despite the evident benefits of implants on mastication as assessed by subjective patient‐based outcomes, the extent of implant overdenture treatment effect on food comminution is not well established. Materials and methods: A randomised clinical trial was carried out with 29 completely edentulous patients divided into two groups. The first group was rehabilitated with a mandibular overdenture retained by two splinted implants with bar‐clip system, while the second group was rehabilitated with a mandibular CD. Both groups also were rehabilitated with maxillary CDs. Masticatory efficiency and patient satisfaction were assessed 3 months after denture insertion. Masticatory efficiency was evaluated through the colorimetric method with the beads as the artificial test‐food. Comparisons for masticatory efficiency and patient satisfaction were performed using Student’s t‐test (α = 0.05). Results: No significant statistical difference was found for masticatory efficiency (p = 0.198). Patient overall satisfaction was significantly higher for the mandibular overdenture (p < 0.001). In addition, mandibular overdenture patients were significantly more satisfied with chewing experience (p < 0.05) and retention of the lower denture (p < 0.005). Conclusion: The results of this study suggest that mandibular overdenture significantly improves chewing experience, although limited effect on masticatory efficiency has been observed.     

Potential role for IL-7 in Fas-mediated T cell apoptosis during HIV infection

IL-7 promotes survival of resting T lymphocytes and induces T cell proliferation in lymphopenic conditions. As elevated IL-7 levels occur in HIV-infected individuals in addition to high Fas expression on T cells and increased sensitivity to Fas-induced apoptosis, we analyzed whether IL-7 has a regulatory role in Fas-mediated T cell apoptosis. We show that IL-7 up-regulates Fas expression on naive and memory T cells through a mechanism that involves translocation of Fas molecules from intracellular compartments to the cell membrane. IL-7 induced the association of Fas with the cytoskeletal component ezrin and a polarized Fas expression on the cell surface. The potential role of IL-7 in Fas up-regulation in vivo was verified in IL-7-treated macaques and in HIV-infected or chemotherapy treated patients by the correlation between serum IL-7 levels and Fas expression on T cells. IL-7 treatment primed T cells for Fas-induced apoptosis in vitro and serum IL-7 levels correlated with the sensitivity of T cells to Fas-induced apoptosis in HIV-infected individuals. Our data suggest an important role for IL-7 in Fas-mediated regulation of T cell homeostasis. Elevated IL-7 levels associated with lymphopenic conditions, including HIV-infection, might participate in the increased sensitivity of T cells for activation-induced apoptosis.     

Nucleophosmin mutations alter its nucleolar localization by impairing G-quadruplex binding at ribosomal DNA

Nucleophosmin (NPM1) is an abundant nucleolar protein implicated in ribosome maturation and export, centrosome duplication and response to stress stimuli. NPM1 is the most frequently mutated gene in acute myeloid leukemia. Mutations at the C-terminal domain led to variant proteins that aberrantly and stably translocate to the cytoplasm. We have previously shown that NPM1 C-terminal domain binds with high affinity G-quadruplex DNA. Here, we investigate the structural determinants of NPM1 nucleolar localization. We show that NPM1 interacts with several G-quadruplex regions found in ribosomal DNA, both in vitro and in vivo. Furthermore, the most common leukemic NPM1 variant completely loses this activity. This is the consequence of G-quadruplex–binding domain destabilization, as mutations aimed at refolding the leukemic variant also result in rescuing the G-quadruplex–binding activity and nucleolar localization. Finally, we show that treatment of cells with a G-quadruplex selective ligand results in wild-type NPM1 dislocation from nucleoli into nucleoplasm. In conclusion, this work establishes a direct correlation between NPM1 G-quadruplex binding at rDNA and its nucleolar localization, which is impaired in the acute myeloid leukemia-associated protein variants.     

Genomic sequence of Spodoptera frugiperda Ascovirus 1a, an enveloped, double-stranded DNA insect virus that manipulates apoptosis for viral reproduction

Ascoviruses (family Ascoviridae) are double-stranded DNA viruses with circular genomes that attack lepidopterans, where they produce large, enveloped virions, 150 by 400 nm, and cause a chronic, fatal disease with a cytopathology resembling that of apoptosis. After infection, host cell DNA is degraded, the nucleus fragments, and the cell then cleaves into large virion-containing vesicles. These vesicles and virions circulate in the hemolymph, where they are acquired by parasitic wasps during oviposition and subsequently transmitted to new hosts. To develop a better understanding of ascovirus biology, we sequenced the genome of the type species Spodoptera frugiperda ascovirus 1a (SfAV-1a). The genome consisted of 156,922 bp, with a G+C ratio of 49.2%, and contained 123 putative open reading frames coding for a variety of enzymes and virion structural proteins, of which tentative functions were assigned to 44. Among the most interesting enzymes, due to their potential role in apoptosis and viral vesicle formation, were a caspase, a cathepsin B, several kinases, E3 ubiquitin ligases, and especially several enzymes involved in lipid metabolism, including a fatty acid elongase, a sphingomyelinase, a phosphate acyltransferase, and a patatin-like phospholipase. Comparison of SfAV-1a proteins with those of other viruses showed that 10% were orthologs of Chilo iridescent virus proteins, the highest correspondence with any virus, providing further evidence that ascoviruses evolved from a lepidopteran iridovirus. The SfAV-1a genome sequence will facilitate the determination of how ascoviruses manipulate apoptosis to generate the novel virion-containing vesicles characteristic of these viruses and enable study of their origin and evolution.     

Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuringiensis subsp. morrisoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101.

The mosquitocidal properties of Bacillus thuringiensis subsp. israelensis and B. thuringiensis subsp. morrisoni PG-14 are attributable to protein inclusions grouped together within a parasporal body. In both of these strains, the mosquitocidal activity resides in proteins with molecular masses of 27, 72, 128, and 135 kDa. In an attempt to determine the toxicity of each protein, the shuttle vector pHT3101 was used to express the cryIVD gene (encoding the 72-kDa CryIVD protein) from B. thuringiensis subsp. morrisoni in an acrystalliferous mutant of B. thuringiensis subsp. kurstaki. With this system, parasporal inclusions of the 72-kDa protein were obtained that were comparable in size, shape, and toxicity to those produced by parental B. thuringiensis subsp. morrisoni. The inclusions were bar shaped, measured 500 by 300 by 150 nm, and were easily visible with phase-contrast microscopy by 16 h of cell growth. A 50% lethal concentration of 64 ng/ml for these inclusions was determined in bioassays against fourth instars of Culex quinquefasciatus, which was similar to the 50% lethal concentration of 55 ng/ml obtained for the 72-kDa inclusion from B. thuringiensis subsp. israelensis. In contrast, expression of the cryIVD gene in Escherichia coli was very low and only detectable by immunoblot analysis. These results demonstrate that the pHT3101-B. thuringiensis expression system can be used to express the CryIVD protein in quantities and with properties comparable to that obtained with the natural host. This system may prove useful for the expression of other B. thuringiensis proteins and, in particular, for reconstitution experiments with inclusions produced by the mosquitocidal subspecies of B. thuringiensis.     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Molecular Dynamics Simulations Elucidate the Mechanism of Proton Transport in the Glutamate Transporter EAAT3

The uptake of glutamate in nerve synapses is carried out by the excitatory amino acid transporters (EAATs), involving the cotransport of a proton and three Na⁺ ions and the countertransport of a K⁺ ion. In this study, we use an EAAT3 homology model to calculate the pK_a of several titratable residues around the glutamate binding site to locate the proton carrier site involved in the translocation of the substrate. After identifying E374 as the main candidate for carrying the proton, we calculate the protonation state of this residue in different conformations of EAAT3 and with different ligands bound. We find that E374 is protonated in the fully bound state, but removing the Na2 ion and the substrate reduces the pK_a of this residue and favors the release of the proton to solution. Removing the remaining Na⁺ ions again favors the protonation of E374 in both the outward- and inward-facing states, hence the proton is not released in the empty transporter. By calculating the pK_a of E374 with a K⁺ ion bound in three possible sites, we show that binding of the K⁺ ion is necessary for the release of the proton in the inward-facing state. This suggests a mechanism in which a K⁺ ion replaces one of the ligands bound to the transporter, which may explain the faster transport rates of the EAATs compared to its archaeal homologs.