Characterization of LeMir, a Root-Knot Nematode-Induced Gene in Tomato with an Encoded Product Secreted from the Root

A tomato gene that is induced early after infection of tomato (Lycopersicon esculentum Mill.) with root-knot nematodes (Meloidogyne javanica) encodes a protein with 54% amino acid identity to miraculin, a flavorless protein that causes sour substances to be perceived as sweet. This gene was therefore named LeMir (L. esculentum miraculin). Sequence similarity places the encoded protein in the soybean trypsin-inhibitor family (Kunitz). LeMir mRNA is found in root, hypocotyl, and flower tissues, with the highest expression in the root. Rapid induction of expression upon nematode infection is localized to root tips. In situ hybridization shows that LeMir is expressed constitutively in the root-cap and root-tip epidermis. The LeMir protein product (LeMir) was produced in the yeast Pichia pastoris for generation of antibodies. Western-blot analysis showed that LeMir expression is up-regulated by nematode infection and by wounding. LeMir is also expressed in tomato callus tissue. Immunoprint analysis revealed that LeMir is expressed throughout the seedling root, but that levels are highest at the root/shoot junction. Analysis of seedling root exudates revealed that LeMir is secreted from the root into the surrounding environment, suggesting that it may interact with soil-borne microorganisms.Root-knot nematodes (Meloidogyne spp.) are endoparasites of the roots of most cultivated crops and cause significant economic losses worldwide (Sasser, 1980). This group of nematodes has a complex life cycle (Williamson and Hussey, 1996). The infective stage, the second-stage juvenile, is attracted to root tips, where it penetrates the zone of elongation and then migrates intercellularly, first toward the root tip and then up to the developing vascular tissue (Wyss et al., 1992). There the nematode initiates a feeding site, causing the formation of large, multinucleate, metabolically active giant cells (Jones, 1978; Huang, 1985). Nearby cells of the cortex, pericycle, and vascular parenchyma enlarge and divide, forming a root-knot or gall. Initiation of the gall can be seen under the microscope between 12 and 24 h after inoculation. Although tomato (Lycopersicon esculentum Mill.) is an excellent host for these nematodes, some varieties are resistant because of the presence of the dominant gene Mi. The presence of Mi is correlated with the development of a localized necrosis of host cells at the feeding site within 24 h of infection (Dropkin et al., 1969; Paulson and Webster, 1972).Because of the economic importance of the root-knot nematode, the molecular biology of the formation and maintenance of the feeding site has been studied by a number of authors (for review, see Williamson and Hussey, 1996). Several genes have been characterized that are up-regulated in the giant cell and the gall (Goddijn et al., 1993; Niebel et al., 1993, 1995, 1996; Bird and Wilson, 1994; Opperman et al., 1994). However, very little is understood regarding molecular changes that occur in the root early after infection, before giant-cell initiation or induction of the hypersensitive response. Genes induced early after nematode infection could potentially have a role in the defense against nematodes or other root pathogens. Overall, root defense systems are poorly understood compared with shoot systems. Of the cases examined, proteins induced in the root during pathogenesis are similar to antimicrobial proteins found in the shoot, such as chitinase, β-1,3-glucanase, osmotin, and ribosome-inactivating protein (Maraganore et al., 1987; Benhamou et al., 1990, 1993; Neale et al., 1990; Savary and Flores, 1994; Savary et al., 1997).To identify rapidly up-regulated, nematode-induced plant genes with a possible role in defense against nematodes or other root parasites, we developed a technique to obtain synchronously infected root tips and then produced a cDNA library from them (Ho et al., 1992; Lambert and Williamson, 1993). Several genes that are increased in expression by 12 h after nematode infection were identified by differential screening (Williamson et al., 1994; Lambert, 1995). Most of these genes appeared to be equally induced in plants independent of the presence of Mi in the genome. Some have homology to known plant defense genes, including those coding for peroxidase, chitinase, lipoxygenase, and proteinase inhibitors (Lambert, 1995; B. Ferrie and V.M. Williamson, unpublished data). One nematode-induced cDNA, clone 23a, encoded the partial sequence of a protein with high similarity to that of miraculin, a protein isolated from the berries of Richadella dulcifica, a west-African shrub.Miraculin alters human taste perception, converting sour into sweet taste (Theerasilp et al., 1989). Because of its sequence similarity to miraculin, the tomato gene was named LeMir (L. esculentum miraculin). The sequence also shows similarity to the soybean trypsin-inhibitor family. Several members of this family have anti-insect/anti-pathogen activity (Ryan, 1990), suggesting that LeMir may have a role in defense against nematodes or other pathogens/pests. Furthermore, LeMir shows very high similarity to TID91, a gene of unknown function that is highly expressed in tobacco genetic tumors (Fujita et al., 1994). In the present study, we characterized the LeMir cDNA sequence and we present information regarding its expression pattern at the mRNA and protein levels.     

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A method for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography.2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with M_r of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 μmol of cytochrome b, 4 μmol of cytochrome c₁ 7–8 μmol of nonheme iron, corresponding to 3.5–4 μmol of the Rieske iron-sulfur protein, less than 1.0 μmol of ubiquinone and about 60 μmol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2 g of Triton X-100 per g protein.3. Cytochrome b exhibits an α-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. ?10 and +100 mV, at pH 7.0. The absorbance maximum of cytochrome c₁ lies at 553 nm and its half-reduction potential amounts to +250 mV.4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20°C.5. A dissociation constant K′_s of 5.5 mM has been determined for the Triton-solubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas.6. A K′_m value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.     

Differential Inhibition of Vesicular Stomatitis Virus Polypeptide Synthesis by Hypertonic Initiation Block

Synthesis of the vesicular stomatitis virus membrane matrix protein and the glycoprotein is inhibited to a greater extent than the synthesis of the nucleocapsid protein, the nonstructural protein, and the large protein when the rate of peptide chain initiation is reduced by exposure of vesicular stomatitis virus-infected cells to hypertonic medium. It is concluded that the relative sensitivity of individual viral polypeptide synthesis to hypertonic initiation block is independent of the site of synthesis, i.e., whether on membrane-associated or free polyribosomes.     

Why there are no objective values: A critique of ethical intuitionism from an evolutionary point of view

Using concepts of evolutionary game theory, this paper presents a critique of ethical intuitionism, or non-naturalism, in its cognitivist and objectivist interpretation. While epistemological considerations suggest that human rational learning through experience provides no basis for objective moral knowledge, it is argued below that modern evolutionary theory explains why this is so, i.e., why biological organisms do not evolve so as to experience objective preferences and obligations. The difference between the modes of the cognition of objective and of valuative environmental attributes is explained with reference to different modes of natural selection acting on the cognitive apparatus of the organism. The negative implications are pointed out which the observable diversity of intraspecific behavioural adaptations and of cultural values has for the cognitivist, objectivist foundation of ethics. Eventually a non-cognitivist alternative to ethical intuitionism is outlined in terms of empirical authority relations, with the ritualisation of dominance-submission patterns as the evolutionary origin of human charismatic authority.     

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.     

Micropropagation of four cultivars of Saskatoon berry (Amelanchier alnifolia NUTT.)

In vitro culture establishment, shoot proliferation, ex vitro rooting and dormancy breaking of the newly rooted plantlets were examined on Saskatoon berry (Amelanchier alnifolia NUTT.) cultivars Northline, Pembina, Smoky and Thiessen. Shoot-tip explants taken from actively growing plants were better for culture initiation than dormant buds. MS gave the most satisfactory results of the media formulations. Optimal shoot proliferation occurred at 8.8 and 13.3 M BA. Higher BA concentrations caused culture deterioration during long-term maintenance. Auxin treatments significantly stimulated ex vitro rooting of shoots in all cultivars. The best rooting was achieved with IAA/NAA (2.8/1.1 M) mixture. Satisfactory results were also obtained with commercial powder formulation, Rootone F, containing IBA/NAA mixture. Foliar application of BA and GA₄₊₇ was successful in breaking dormancy of newly rooted plantlets. Combinations of these two growth regulators caused formation of axillary shoots and vigorous plant growth. There were significant differences in the cultivar responses to culture conditions and treatments with growth regulators. The best culture establishment and the highest rate of shoot proliferation was observed in cv. Thiessen; the best rooting and the most vigorous post-dormancy growth was recorded in cv. Smoky. Cultivar Northland gave the most erratic responses.Abbreviations BA benzyladenine - cv(s) cultivar(s) - GA gibberellin - IAA indoleacetic acid - IBA indolebutyric acid - NAA naphthaleneacetic acid - MS Murashige & Skoog's medium     

Mediterranean types of β-thalassemia in the German population

Summary Forty -thalassemia genes from unrelated German heterozygotes with no known foreign ancestry were examined using the oligonucleotide technique and DNA restriction analysis, with the aim of determining the contribution of Mediterranean -thalassemia mutations to the prevalence of this trait in the German population. Of the 40 -thalassemia genes, 26 were identified as Mediterranean types (20 39 nonsense, 3 IVS2 nt 110, 2 IVS2 nt1, 1 IVS1 ntl GA). The geographic distribution of the birthplaces of the probands' grandparents revealed no difference in the proportion of Mediterranean and unidentified -thalassemia genes in the west and the north of Germany.     

Discovery of selective 2,4-diaminoquinazoline toll-like receptor 7 (TLR 7) agonists

The discovery of a novel series of highly potent quinazoline TLR 7/8 agonists is described. The synthesis and structure–activity relationship is presented. Structural requirements and optimization of this series toward TLR 7 selectivity afforded the potent agonist 48. Pharmacokinetic and pharmacodynamic studies highlighted 48 as an orally available endogenous interferon (IFN-α) inducer in mice.