Crystal structure at 1.8 A resolution and identification of active site residues of Sulfolobus solfataricus peptidyl-tRNA hydrolase

The 3-D structure of the peptidyl-tRNA hydrolase from the archaea Sulfolobus solfataricus has been solved at 1.8 A resolution. Homologues of this enzyme are found in archaea and eucarya. Bacteria display a different type of peptidyl-tRNA hydrolase that is also encountered in eucarya. In solution, the S. solfataricus hydrolase behaves as a dimer. In agreement, the crystalline structure of this enzyme indicates the formation of a dimer. Each protomer is made of a mixed five-stranded beta-sheet surrounded by two groups of two alpha-helices. The dimer interface is mainly formed by van der Waals interactions between hydrophobic residues belonging to the two N-terminal alpha1 helices contributed by two protomers. Site-directed mutagenesis experiments were designed for probing the basis of specificity of the archaeal hydrolase. Among the strictly conserved residues within the archaeal/eucaryal peptidyl-tRNA hydrolase family, three residues, K18, D86, and T90, appear of utmost importance for activity. They are located in the N-part of alpha1 and in the beta3-beta4 loop. K18 and D86, which form a salt bridge, might play a role in the catalysis thanks to their acid and basic functions, whereas the OH group of T90 could act as a nucleophile. These observations clearly distinguish the active site of the archaeal/eucaryal hydrolases from that of the bacterial/eucaryal ones, where a histidine is believed to serve as the catalytic base.     

Responses to K deficiency and waterlogging interact via respiratory and nitrogen metabolism

K deficiency and waterlogging are common stresses that can occur simultaneously and impact on crop development and yield. They are both known to affect catabolism, with rather opposite effects: inhibition of glycolysis and higher glycolytic fermentative flux, respectively. But surprisingly, the effect of their combination on plant metabolism has never been examined precisely. Here, we applied a combined treatment (K availability and waterlogging) to sunflower (Helianthus annuus L.) plants under controlled greenhouse conditions and performed elemental quantitation, metabolomics, and isotope analyses at different sampling times. Whereas separate K deficiency and waterlogging caused well‐known effects such as polyamines production and sugar accumulation, respectively, waterlogging altered K‐induced respiration enhancement (via the C₅‐branched acid pathway) and polyamine production, and K deficiency tended to suppress waterlogging‐induced accumulation of Krebs cycle intermediates in leaves. Furthermore, the natural ¹⁵N/¹⁴N isotope composition (δ¹⁵N) in leaf compounds shows that there was a change in nitrate circulation, with less nitrate influx to leaves under low K availablity combined with waterlogging and more isotopic dilution of lamina nitrates under high K. Our results show that K deficiency and waterlogging effects are not simply additive, reshape respiration as well as nitrogen metabolism and partitioning, and are associated with metabolomic and isotopic biomarkers of potential interest for crop monitoring.     

Cd(II), Pb(II) and Zn(II) ions regulate expression of the metal-transporting P-type ATPase ZntA in Escherichia coli

Feeding bioassay results established that the soybean cysteine proteinase inhibitor N (soyacystatin N, scN) substantially inhibits growth and development of western corn rootworm (WCR), by attenuating digestive proteolysis [Zhao, Y. et al. (1996) Plant Physiol. 111, 1299-1306]. Recombinant scN was more inhibitory than the potent and broad specificity cysteine proteinase inhibitor E-64. WCR digestive proteolytic activity was separated by mildly denaturing SDS-PAGE into two fractions and in-gel assays confirmed that the proteinase activities of each were largely scN-sensitive. Since binding affinity to the target proteinase [Koiwa, H. et al. (1998) Plant J. 14, 371-380] governs the effectiveness of scN as a proteinase inhibitor and an insecticide, five peptides (28-33 kDa) were isolated from WCR gut extracts by scN affinity chromatographic separation. Analysis of the N-terminal sequence of these peptides revealed similarity to a cathepsin L-like cysteine proteinase (DvCAL1, Diabrotica virgifera virgifera cathepsin L) encoded by a WCR cDNA. Our results indicate that cathepsin L orthologs are pivotal digestive proteinases of WCR larvae, and are targets of plant defensive cystatins (phytocystatins), like scN.     

Ghost mtDNA haplotypes generated by fortuitous NUMTs can deeply disturb infra‐specific genetic diversity and phylogeographic pattern

Nuclear copies of mitochondrial DNA (NUMTs or mitochondrial pseudogenes) are known to impede the detection of interspecific genetic diversity. But the effect of these artifacts on phylogeographic reconstruction remains under evaluated. In this study, we analysed a set of 115 sequences of a fragment of the cytochrome c oxidase subunit I gene (COI) of Monochamus galloprovincialis (Coleoptera, Cerambycidae) for which overlapping signals in sequencing electropherograms were observed. Comparison of full and corrected ‘ambiguities‐free’ data sets reveals the prevalence of numerous supernumerary haplotypes that deeply affect genetic diversity indices and phylogeographic patterns of this species. Slightly divergent pseudogenes were recovered in 49 of the 115 sequences. These results highlight the potential misdetection of NUMTs using current control methods and the consequences on phylogeographic structure. To test the frequency of unintended amplification of NUMTs, a cloning was performed on 15 individuals. An average of 3.72 and a maximum of six paralogous sequences with different levels of divergence were identified among individual cloned. Within individual pairwise distance between paralogs raised 1.4%. This work calls for awareness to the presence of undetected NUMTs within mitochondrial data sets, especially at infra‐specific level.     

Inactivation of the 3'-5' exonuclease of the replicative T4 DNA polymerase allows translesion DNA synthesis at an abasic site

Here, we have investigated the consequences of the loss of proof-reading exonuclease function on the ability of the replicative T4 DNA polymerase (gp43) to elongate past a single abasic site located on model DNA substrates. Our results show that wild-type T4 DNA polymerase stopped at the base preceding the lesion on two linear substrates having different sequences, whereas the gp43 D219A exonuclease-deficient mutant was capable of efficient bypass when replicating the same substrates. The structure of the DNA template did not influence the behavior of the exonuclease-proficient or deficient T4 DNA polymerases. In fact, when replicating a damaged "minicircle" DNA substrate constructed by circularizing one of the linear DNA, elongation by wild-type enzyme was still completely blocked by the abasic site, while the D219A mutant was capable of bypass. During DNA replication, the T4 DNA polymerase associates with accessory factors whose combined action increases the polymerase-binding capacity and processivity, and could modulate the behavior of the enzyme towards an abasic site. We thus performed experiments measuring the ability of wild-type and exonuclease-deficient T4 DNA polymerases, in conjunction with these replicative accessory proteins, to perform translesion DNA replication on linear or circular damaged DNA substrates. We found no evidence of either stimulation or inhibition of the bypass activities of the wild-type and exonuclease-deficient forms of T4 DNA polymerase following addition of the accessory factors, indicating that the presence or absence of the proof-reading activity is the major determinant in dictating translesion synthesis of an abasic site by T4 DNA polymerase.     

Maternal Short-Chain Fructooligosaccharide Supplementation Influences Intestinal Immune System Maturation in Piglets

Peripartum nutrition is crucial for developing the immune system of neonates. We hypothesized that maternal short-chain fructooligosaccharide (scFOS) supplementation could accelerate the development of intestinal immunity in offspring. Thirty-four sows received a standard or a scFOS supplemented diet (10 g scFOS/d) for the last 4 weeks of gestation and the 4 weeks of lactation. Colostrum and milk immunoglobulins (Ig) and TGFβ1 concentrations were evaluated on the day of delivery and at d 6 and d 21 postpartum. Piglet intestinal structure, the immunologic features of jejunal and ileal Peyer''s patches, and mesenteric lymph node cells were analysed at postnatal d 21. Short-chain fatty acid concentrations were measured over time in the intestinal contents of suckling and weaned piglets. Colostral IgA (P<0.05) significantly increased because of scFOS and TGFβ1 concentrations tended to improve (P<0.1). IFNγ secretion by stimulated Peyer''s patch and mesenteric lymph node cells, and secretory IgA production by unstimulated Peyer''s patch cells were increased (P<0.05) in postnatal d 21 scFOS piglets. These differences were associated with a higher proportion of activated CD25+CD4α+ T cells among the CD4+ helper T lymphocytes (P<0.05) as assessed by flow cytometry. IFNγ secretion was positively correlated with the population of activated T lymphocytes (P<0.05). Total short-chain fatty acids were unchanged between groups during lactation but were higher in caecal contents of d 90 scFOS piglets (P<0.05); specifically propionate, butyrate and valerate. In conclusion, we demonstrated that maternal scFOS supplementation modified the intestinal immune functions in piglets in association with increased colostral immunity. Such results underline the key role of maternal nutrition in supporting the postnatal development of mucosal immunity.     

Hepatitis B virus entry into HepG2‐NTCP cells requires clathrin‐mediated endocytosis

Hepatitis B virus (HBV) is a leading cause of cirrhosis and hepatocellular carcinoma worldwide, with 250 million individuals chronically infected. Many stages of the HBV infectious cycle have been elucidated, but the mechanisms of HBV entry remain poorly understood. The identification of the sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor and the establishment of NTCP‐overexpressing hepatoma cell lines susceptible to HBV infection opens up new possibilities for investigating these mechanisms. We used HepG2‐NTCP cells, and various chemical inhibitors and RNA interference (RNAi) approaches to investigate the host cell factors involved in HBV entry. We found that HBV uptake into these cells was dependent on the actin cytoskeleton and did not involve macropinocytosis or caveolae‐mediated endocytosis. Instead, entry occurred via the clathrin‐mediated endocytosis pathway. HBV internalisation was inhibited by pitstop‐2 treatment and RNA‐mediated silencing (siRNA) of the clathrin heavy chain, adaptor protein AP‐2 and dynamin‐2. We were able to visualise HBV entry in clathrin‐coated pits and vesicles by electron microscopy (EM) and cryo‐EM with immunogold labelling. These data demonstrating that HBV uses a clathrin‐mediated endocytosis pathway to enter HepG2‐NTCP cells increase our understanding of the complete HBV life cycle.     

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

Rho-kinase and myosin-II control phagocytic cup formation during CR,but not FcgammaR,phagocytosis

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at CR phagosomes. To better understand the role of Rho in CR phagocytosis, we tested the idea that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. We found that inhibition of the Rho --> ROK --> myosin-II pathway caused a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which led to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, was independent of Rho or ROK activity and was only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, to our knowledge, this is the first demonstration of a role for myosin-II in CR phagocytosis.     

Crystallization and preliminary x-ray analysis of Escherichia coli peptidyl-tRNA hydrolase

Peptidyl-tRNA hydrolase from Escherichia coli, a monomer of 21 kDa, was overexpressed from its cloned gene pth and crystallized by using polyethylene glycol as precipitant. The crystals are orthorhombic and have unit cell parameters a = 47.24 Å, b = 63.59 Å, and c = 62.57 Å. They belong to space group P2₁2₁2₁ and diffract to better than 1.2 Å resolution. The structure is being solved by multiple isomorphous replacement. © 1997 Wiley-Liss Inc.