Rapid Pathogen-Induced Apoptosis: A Mechanism Used by Dendritic Cells to Limit Intracellular Replication of Legionella pneumophila

Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.     

A new species of Bembidion ( Ecuadion) from Ecuador (Coleoptera,Carabidae, Bembidiini), with a key to members of the georgeballi species group

A new species of ground beetle, Bembidion ricei, is described from the Andes mountains of Ecuador east of Quito. It belongs to the georgeballi species group of subgenus Ecuadion, and is most similar to Bembidion georgeballi. A key to the species of the group is provided.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Paraiotonchium muscadomesticae n. sp. (Tylenchida: lotonchiidae) a Parasite of the House Fly (Musca domestica) in Brazil and a Key to Species of the Genus Paraiotonchium

Paraiotonchium muscadomesticae n. sp., a parasite of the house fly, Musca domestica L., is described and illustrated from material collected in Brazil. The life cycle of P. muscadomesticae is similar to that of P. autumnale (Nickle), consisting of alternating gamogenetic and parthenogenetic generations. Paraiotonchium muscadomesticae n. sp. can be distinguished from P. nicholasi Slobodyanyuk, P. autumnale (Nickle) Slobodyanyuk, and P. crassirostris (Yatham &Rao) Siddiqi by the shorter body length of young heterosexual females, 652 g.m (530-709) for P. muscadomesticae compared to 750 μm or more (801-1,050) for the others. Paraiotonchium muscadomesticae is close to P. nicholosi but differs from it by V ratio and spicule length (V = 80-84; spicule = 16-21 p.m in P. muscadomesticae compared to V = 73-78; spicule = 25-35 μm in P. nicholasi). Paraiotonchium muscadomesticae and P. nicholasi differ from all species of this genus by the absence of a bursa on males of these two species.     

Characterization of WbpB, WbpE, and WbpD and Reconstitution of a Pathway for the Biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic Acid in Pseudomonas aeruginosa

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His₆-WbpB and His₆-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.Gram-negative organisms such as Pseudomonas aeruginosa produce lipopolysaccharide (LPS)⁴ as an essential component of the outer leaflet of the outer membrane. LPS can be conceptually divided into three parts: lipid A, which anchors LPS into the membrane; core oligosaccharide, which contributes to membrane stability; and the O-antigen, which is a polysaccharide that extends away from the cell surface. In P. aeruginosa, two types of O-antigen are observed: A-band O-antigen, which is common to most strains, and B-band O-antigen, which is variable and therefore used as the basis of the International Antigenic Typing Scheme (1). P. aeruginosa serotypes O2, O5, O16, O18, and O20 collectively belong to serogroup O2, because they all share common backbone sugar structures in their O-antigen repeat units consisting of two di-N-acetylated uronic acids and one 2-acetamido-2,6-dideoxy-d-galactose (N-acetyl-d-fucosamine). The minor structural variations in the O-antigen repeat units that differentiate this serogroup into five serotypes are: the type of glycosidic linkage between O-units (alpha versus beta) that is formed by the O-antigen polymerase (Wzy), isomers present (d-mannuronic or l-guluronic acid), and acetyl group substituents (2–4). The B-band O-antigen of P. aeruginosa PAO1 (serotype O5) contains a repeating trisaccharide of 2-acetamido-3-acetamidino-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAmA), 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA), and 2-acetamido-2,6-dideoxy-d-galactose (3).The biosynthesis of the two mannuronic acid derivatives has yet to be fully understood and has been the subject of investigation by our group. To produce UDP-d-ManNAc3NAcA, a five-step pathway has been proposed (Fig. 1) that requires the products of five genes localized to the B-band O-antigen biosynthesis cluster (5). The O-antigen biosynthesis cluster was shown to be identical for all serotypes within serogroup O2, which further underscores the high similarity between these serotypes (5). The five genes, including wbpA, wbpB, wbpE, wbpD, and wbpI, have been shown to be essential for B-band LPS biosynthesis, because knockout mutants of each of these genes are deficient in B-band O-antigen (6–8). Homologs of all five of the proteins required for the UDP-d-ManNAc3NAcA biosynthesis pathway are conserved in other bacterial pathogens, including Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Cross-complementation of P. aeruginosa knockout mutants lacking wbpA, wbpB, wbpE, wbpD, or wbpI with the homologues from B. pertussis could fully restore LPS production in the P. aeruginosa LPS mutants, suggesting that the genes from B. pertussis are functional homologs of the wbp genes (7). Homologs of these genes could be identified in diverse bacterial species, demonstrating the importance of UDP-d-ManNAc3NAcA biosynthesis beyond its role in P. aeruginosa (7).Open in a separate windowFIGURE 1.Proposed pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa PAO1. The full names of the sugars are as follows: GlcNAc, 2-acetamido-2-deoxy-d-glucose; GlcNAcA, 2-acetamido-2-deoxy-d-glucuronic acid; 3-keto-d-GlcNAcA, 2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid; GlcNAc3NA, 2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid; GlcNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid; ManNAc3NAcA, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid. Adapted from Ref. 8.The first enzyme of the UDP-d-ManNAc3NAcA biosynthesis pathway, WbpA, is a 6-dehydrogenase that converts UDP-2-acetamido-2-deoxy-d-glucose (N-acetyl-d-glucosamine; UDP-d-GlcNAc) to UDP-2-acetamido-2-deoxy-d-glucuronic acid (N-acetyl-d-glucosaminuronic acid, UDP-d-GlcNAcA) using NAD⁺ as a coenzyme (9) (Fig. 1). Following this, the second step in UDP-d-ManNAc3NAcA biosynthesis is proposed to be an oxidation reaction catalyzed by WbpB, forming UDP-2-acetamido-2-deoxy-d-ribo-hex-3-uluronic acid (3-keto-d-GlcNAcA), which in turn is used as the substrate for transamination by WbpE, creating UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid (d-GlcNAc3NA).This residue is thought to be the substrate for WbpD, a putative N-acetyltransferase of the hexapeptide acyltransferase superfamily (10) that requires acetyl-CoA as a co-substrate (8). WbpD has been proposed to synthesize UDP-2,3-diacetamido-2,3-dideoxy-d-glucuronic acid (UDP-d-GlcNAc-3NAcA), which is utilized in the B-band O-antigen of P. aeruginosa serotype O1. In P. aeruginosa serogroup O2, the UDP-d-GlcNAc3NAcA is then epimerized by WbpI to create the UDP-d-ManNAc3NAcA required for incorporation into B-band LPS (11). A derivative of UDP-d-ManNAc3NAcA is also used in the synthesis of B-band O-antigen of P. aeruginosa serogroup O2. UDP-d-ManNAc3NAmA is thought to be produced through additional modification of UDP-d-ManNAc3NAcA via the action of WbpG, an amidotransferase, which has also been demonstrated to be essential for the production of B-band O-antigen (12, 13).In the current study, our aim was to define the function of WbpB, WbpE, and WbpD, because only genetic evidence has previously been given for the involvement of wbpB and wbpE (7), and the reaction catalyzed by WbpD could not be demonstrated due to the unavailability of its presumed substrate, UDP-d-GlcNAc3NA (8). The functional characterization of these proteins is also important for understanding LPS biosynthesis in B. pertussis, because the genes in the LPS locus of this species, wlbA, wlbC, and wlbB, could cross-complement knockouts of wbpB, wbpE, and wbpD, respectively, when expressed in P. aeruginosa PAO1 (7). Furthermore, these three proteins form a cassette for the generation of C-3 N-acetylated hexoses and may be important for the biosynthesis of a variety of other sugars. Capillary electrophoresis and MALDI-TOF mass spectrometry were used to analyze reaction mixtures of WbpB and WbpE and showed that the expected products were produced only when both enzymes were present together. Achieving the enzymatic synthesis of the product of both enzymes, which was demonstrated to be UDP-d-GlcNAc3NA by ¹H NMR spectroscopy, was a key breakthrough, because this rare sugar has never before been produced by any means. UDP-d-GlcNAc3NA was also essential for use as the substrate of WbpD, which not only allowed us to determine the enzymatic activity of this protein but also allowed the enzymatic synthesis of UDP-d-GlcNAc3NAcA to be achieved as well. Although this sugar had previously been produced through a 17-step chemical synthesis (11, 14), the 4-step concurrent enzymatic reaction demonstrates the advantage of linking chemistry with biology and represents a significant saving of both time and reagents as compared with chemical synthesis. Finally, our data also showed the success in reconstituting in vitro the 5-step pathway for the biosynthesis of UDP-d-ManNAc3NAcA in P. aeruginosa.     

Calmodulin Binds to Extracellular Sites on the Plasma Membrane of Plant Cells and Elicits a Rise in Intracellular Calcium Concentration

Calmodulin (CaM) is a highly conserved intracellular calcium sensor. In plants, CaM also appears to be present in the apoplasm, and application of exogenous CaM has been shown to influence a number of physiological functions as a polypeptide signal; however, the existence and localization of its corresponding apoplasmic binding sites remain controversial. To identify the site(s) of action, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection at the surface of plant cells. Using this approach, we show that QD-CaM binds selectively to sites on the outer surface of the plasma membrane, which was further confirmed by high resolution transmission electron microscopy. Measurements of Ca²⁺ fluxes across the plasma membrane, using ion-selective microelectrodes, demonstrated that exogenous CaM induces a net influx into protoplasts. Consistent with these flux studies, calcium-green-dextran and FRET experiments confirmed that applied CaM/QD-CaM elicited an increase in cytoplasmic Ca²⁺ levels. These results support the hypothesis that apoplasmic CaM can act as a signaling agent. These findings are discussed in terms of CaM acting as an apoplasmic peptide ligand to mediate transmembrane signaling in the plant kingdom.Calmodulin (CaM)² is a conserved multifunctional calcium sensor that mediates intracellular Ca²⁺ signaling and regulates diverse cellular processes by interacting with calmodulin-binding proteins (1–3). Interestingly, in both animals and plants, CaM may also act as an extracellular agent to regulate physiological events (4). Consistent with this notion, extracellular CaM has been detected within the cell walls of a broad range of plant species (4, 5).Functional studies have established that exogenously applied CaM can stimulate the proliferation of suspension-cultured plant cells (6) as well as affect intracellular activities of heterotrimeric G proteins and phospholipases in protoplasts (7, 8). Based on these findings, it has been proposed that, in plants, extracellular CaM may function as a signaling agent involved in the regulation of cell growth and development (4). However, as a 17-kDa hydrophilic protein, exogenously applied CaM could well be retrieved from the apoplasmic space and then exert its effects on components within the cytoplasm. Evidence against this hypothesis was provided by studies with Arabidopsis thaliana suspension-cultured cells in which it was shown that 24 h of incubation in exogenous CaM did not result in protein uptake or degradation (4).To exert an effect from the apoplasm, it would seem logical to assume that a protein(s) within the plant plasma membrane would have a CaM-binding site exposed to the apoplasm. Although a number of studies have addressed the molecular mechanism(s) by which extracellular CaM might act as a signal (6, 9) and attempts have been made to identify extracellular CaM-binding proteins (4, 6), currently there is no direct evidence in support of the hypothesis that specific CaM-binding sites exist at the surface of plant cells.To address this question, a CaM-conjugated quantum dot (QD) system was employed for single molecule level detection (10–13) at the surface of plant cells. These nanoparticles have several advantages over conventional fluorophores for light microscopic imaging, including their higher brightness and photostability (14, 15). In addition, because of their electron dense nature, QDs can be used for single labeling studies at the transmission electron microscope level (16, 17). Using this QD-CaM system, we demonstrate that QD-CaM binds selectively to sites on the outer surface of the plant plasma membrane. We also show by three independent methods that applied CaM can modulate Ca²⁺ fluxes across the plasma membrane, leading to alterations in cytoplasmic Ca²⁺ status. These findings support the hypothesis that, in plants, apoplasmic CaM can act as a signaling agent.     

Shifts in the Distribution of Mass Densities Is a Signature of Caloric Restriction in Caenorhabditis elegans

Although the starvation response of the model multicellular organism Caenorhabditis elegans is a subject of much research, there is no convenient phenotypic readout of caloric restriction that can be applicable to large numbers of worms. This paper describes the distribution of mass densities of populations of C. elegans, from larval stages up to day one of adulthood, using isopycnic centrifugation, and finds that density is a convenient, if complex, phenotypic readout in C. elegans. The density of worms in synchronized populations of wildtype N2 C. elegans grown under standard solid-phase culture conditions was normally distributed, with distributions peaked sharply at a mean of 1.091 g/cm³ for L1, L2 and L3 larvae, 1.087 g/cm³ for L4 larvae, 1.081 g/cm³ for newly molted adults, and 1.074 g/cm³ at 24 hours of adulthood. The density of adult worms under starvation stress fell well outside this range, falling to a mean value of 1.054 g/cm³ after eight hours of starvation. This decrease in density correlated with the consumption of stored glycogen in the food-deprived worms. The density of the worms increased when deprived of food for longer durations, corresponding to a shift in the response of the worms: worms sacrifice their bodies by retaining larvae, which consume the adults from within. Density-based screens with the drug Ivermectin on worms cultured on single plates resulted in a clear bimodal (double-peaked) distribution of densities corresponding to drug exposed and non-exposed worms. Thus, measurements of changes in density could be used to conduct screens on the effects of drugs on several populations of worms cultured on single plates.     

The Mechanism of Onset of the Stationary Phase in Euglena gracilis Grown with 10 mM Succinate: Intracellular pH Values*

SYNOPSIS. When Euglena gracilis were grown with 10mM succinate at pH 3.5 the extracellular pH averaged 3.62 and the cultures had produced 6 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 30 μliters/10⁶ cells/hr. Total protein and dry weights per cell remained constant during the logarithmic phase and began to decline when the late logarithmic phase was reached. Added succinate caused the cultures in stationary phase to commence logarithmic growth once more. Onset of the stationary phase in cultures grown at pH 3.5 was due to depletion of succinate. When cultures were grown at pH 6.9 the extracellular pH averaged 7.62 and the cultures produced 3 × 10⁵ cells/ml when the stationary phase began. Oxygen consumption values reached a maximum of 20 μliters/10⁶ cells/hr during the logarithmic phase. The decline in total protein and dry weights per cell began at the beginning of the logarithmic phase and continued into the stationary phase of growth. Cultures grown at pH 3.5 should produce a larger number of cells/ml than cultures grown at pH 6.9 if the cells are responding to the unionized moiety of succinate and not the ionized moiety. At pH 3.5 83% of the succinate is unionized, whereas at pH 6.9 0.20% of the succinate is unionized. The onset of the stationary phase in cultures grown at pH 3.5 and pH 6.9 is due to lack of an adequate amount of extracellular unionized succinate. Intracellular pH values were determined in cultures grown at pH 6.9 using the weak acid DMO (5.5-dimethyl-2,4-oxazolidinedione). As the extracellular pH increased from 6.90 to 7.62, the intracellular pH increased from 5.89 to 6.89. As the extracellular pH increased from 7.62 to 8.44, the intracellular pH increased from 6.89 to 7.50.     

Contribution of Malic Enzyme, Pyruvate Kinase, Phosphoenolpyruvate Carboxylase, and the Krebs Cycle to Respiration and Biosynthesis and to Intracellular pH Regulation during Hypoxia in Maize Root Tips Observed by Nuclear Magnetic Resonance Imaging and Gas Chromatography-Mass Spectrometry

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by ¹³C incorporation from [1-¹³C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. ¹³C-Nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.     

Comparison of Crystal Field Dependent and Independent Methods to Analyse Lanthanide Induced NMR Shifts in Axially Symmetric Complexes. Part II: Systems with a C4 Symmetry Axis

Analysis of the LIS data for several series of Ln³⁺ complexes of C₄ symmetry in terms of structural changes, crystal-field effects and/or variation of hyperfine constants along the lanthanide series was undertaken using a combination of the two-nuclei and three-nuclei techniques together with the classical onenucleus technique. Isostructurality of whole series of complexes, with changes of the F_i, and B₀² parameters, was clearly defined for the complexes of L by the combination of the two first methods. Small changes, involving the three F_i, G_i and B₀² parameters, are observed for the series of complexes of L-L4, using the three data plotting methods. Some of the plots according to the two- and three-nuclei methods are accidentally linear, without necessarily implying isostructurality of the complexes, as they involve parameters, which may be insensitive to any small structural changes occurring in these systems. These parameter variations could result from a magnification, by the present graphical analysis, of the breaks expected from the gradual structural changes along the series due to the lanthanide contraction. The α and β parameters of the three-nuclei method are not diagnostic of the type of structures the complexes have in solution, due to their very indirect dependence on the geometric factors.     

Expression of Tobacco Carbonic Anhydrase in the C4 Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO2-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C₄ dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO₂ assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O₂ concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O₂ levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O₂ concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C₄ CO₂-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO₂ for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.     

Structure of a Viral Cap-independent Translation Element That Functions via High Affinity Binding to the eIF4E Subunit of eIF4F

RNAs of many positive strand RNA viruses lack a 5′ cap structure and instead rely on cap-independent translation elements (CITEs) to facilitate efficient translation initiation. The mechanisms by which these RNAs recruit ribosomes are poorly understood, and for many viruses the CITE is unknown. Here we identify the first CITE of an umbravirus in the 3′-untranslated region of pea enation mosaic virus RNA 2. Chemical and enzymatic probing of the ∼100-nucleotide PEMV RNA 2 CITE (PTE), and mutagenesis revealed that it forms a long, bulged helix that branches into two short stem-loops, with a possible pseudoknot interaction between a C-rich bulge at the branch point and a G-rich bulge in the main helix. The PTE inhibited translation in trans, and addition of eIF4F, but not eIFiso4F, restored translation. Filter binding assays revealed that the PTE binds eIF4F and its eIF4E subunit with high affinity. Tight binding required an intact cap-binding pocket in eIF4E. Among many PTE mutants, there was a strong correlation between PTE-eIF4E binding affinity and ability to stimulate cap-independent translation. We conclude that the PTE recruits eIF4F by binding eIF4E. The PTE represents a different class of translation enhancer element, as defined by its structure and ability to bind eIF4E in the absence of an m⁷G cap.Regulation of translation occurs primarily at the initiation step. This involves recognition of the 5′ m⁷G(5′)ppp(5′)N cap structure on the mRNA by initiation factors, which recruit the ribosome to the 5′-end of the mRNA (1–5). The 5′ cap structure and the poly(A) tail are necessary for efficient recruitment of initiation factors on eukaryotic mRNAs (3, 6–8). The cap is recognized by the eIF4E subunit of eukaryotic translation initiation factor complex eIF4F (or the eIFiso4E subunit of eIFiso4F in higher plants). The poly(A) tail is recognized by poly(A)-binding protein. In plants, eIF4F is a heterodimer consisting of eIF4E and eIF4G, the core scaffolding protein to which the other factors bind. eIF4A, an ATPase/RNA helicase, interacts with eIF4F but is not part of the eIF4F heterodimer (9, 10). For translation initiation, the purpose of eIF4E is to bring eIF4G to the capped mRNA. eIF4G then recruits the 43 S ternary ribosomal complex via interaction with eIF3.The RNAs of many positive sense RNA viruses contain a cap-independent translation element (CITE)³ that allows efficient translation in the absence of a 5′ cap structure (11–13). In animal viruses and some plant viruses, the CITE is an internal ribosome entry site (IRES) located upstream of the initiation codon. Most viral IRESes neither interact with nor require eIF4E, because they lack the m⁷GpppN structure, which, until this report, was thought to be necessary for mRNA to bind eIF4E with high affinity (3, 14). Translation initiation efficiency of mRNA is also influenced by the length of, and the degree of secondary structure in the 5′ leader (15–17).Many uncapped plant viral RNAs harbor a CITE in the 3′-UTR that confers highly efficient translation initiation at the 5′-end of the mRNA (18–22). These 3′ CITEs facilitate ribosome entry and apparently conventional scanning at the 5′-end of the mRNA (17, 23, 24). A variety of unrelated structures has been found to function as 3′ CITEs, suggesting that they recruit the ribosome by different interactions with initiation factors (13).The factors with which a plant CITE interacts to recruit the ribosome have been identified for only a potyvirus, a luteovirus, and a satellite RNA. The 143-nt 5′-UTR CITE of the potyvirus, tobacco etch virus is an IRES that functions by binding of its AU-rich pseudoknot structure with eIF4G (25). It binds eIF4G with up to 30-fold greater affinity than eIFiso4G and does not require eIF4E for IRES activity. In addition to RNA elements, the genome-linked viral protein (VPg) of potyviruses may participate in cap-independent translation initiation by interacting with the eIF4E and eIFiso4E subunits of eIF4F and eIFiso4F, respectively (26–31). In contrast, the 130-nt cap-independent translation enhancer domain (TED) in the 3′-UTR of satellite tobacco necrosis virus (STNV) RNA forms a long bulged stem-loop, which interacts strongly with both eIF4F and eIFiso4F and weakly with their eIF4E and eIFiso4E subunits (32), suggesting that the TED requires the full eIF4F or eIFiso4F for a biologically relevant interaction. Barley yellow dwarf luteovirus (BYDV) and several other viruses, have a different structure, called a BYDV-like CITE (BTE), in the 3′-UTR. The BTE is characterized by a 17-nt conserved sequence incorporated in a structure with a variable number of stem-loops radiating from a central junction (20, 33, 34). It requires and binds the eIF4G subunit of eIF4F and does not bind free eIF4E, eIFiso4E, or eIFiso4G, although eIF4E slightly enhances the BTE-eIF4G interaction (35). Other 3′ CITEs have been identified, but the host factors with which they interact are unknown.Here we describe unprecedented factor interactions of a CITE found in an umbravirus and a panicovirus. Umbraviruses show strong similarity to the Luteovirus and Dianthovirus genera in (i) the sequence of the replication genes encoded by ORFs 1 and 2, (ii) the predicted structure of the frameshift signals required for translation of the RNA-dependent RNA polymerase from ORF 2 (36, 37), (iii) the absence of a poly(A) tail, and (iv) the lack of a 5′ cap structure (37, 38). Umbraviruses are unique in that they encode no coat protein. For the umbravirus pea enation mosaic virus 2 (PEMV-2), the coat protein is provided by PEMV-1, an enamovirus (39). Uncapped PEMV-2 RNA (PEMV RNA 2), transcribed in vitro, is infectious in pea (Pisum sativa),⁴ indicating it must be translated cap-independently. The 3′-UTRs of some umbraviruses such as Tobacco bushy top virus and Groundnut rosette virus harbor sequences resembling BYDV-like CITEs (BTE).⁵ However, no BTE is apparent in the 3′-UTR of PEMV RNA 2. In this report we identify a different class of CITE in the 705-nt long 3′-UTR of PEMV RNA 2, determine its secondary structure, which may include an unusual pseudoknot, and we show that, unlike any other natural uncapped RNA, it has a high affinity for eIF4E, which is necessary to facilitate cap-independent translation.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase : IV. Fusicoccin Induces the Association between the Plasma Membrane H+-ATPase and the Fusicoccin Receptor

Different approaches were utilized to investigate the mechanism by which fusicoccin (FC) induces the activation of the H⁺-ATPase in plasma membrane (PM) isolated from radish (Raphanus sativus L.) seedlings treated in vivo with (FC-PM) or without (C-PM) FC. Treatment of FC-PM with different detergents indicated that PM H⁺-ATPase and the FC-FC-binding-protein (FCBP) complex were solubilized to a similar extent. Fractionation of solubilized FC-PM proteins by a linear sucrose-density gradient showed that the two proteins comigrated and that PM H⁺-ATPase retained the activated state induced by FC. Solubilized PM proteins were also fractionated by a fast-protein liquid chromatography anion-exchange column. Comparison between C-PM and FC-PM indicated that in vivo treatment of the seedlings with FC caused different elution profiles; PM H⁺-ATPase from FC-PM was only partially separated from the FC-FCBP complex and eluted at a higher NaCl concentration than did PM H⁺-ATPase from C-PM. Western analysis of fast-protein liquid chromatography fractions probed with an anti-N terminus PM H⁺-ATPase antiserum and with an anti-14–3-3 antiserum indicated an FC-induced association of FCBP with the PM H⁺-ATPase. Analysis of the activation state of PM H⁺-ATPase in fractions in which the enzyme was partially separated from FCBP suggested that the establishment of an association between the two proteins was necessary to maintain the FC-induced activation of the enzyme.