Analysis of lectin binding by Bradyrhizobium japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin

A procedure was developed to assess the ability of wild-type and mutant strains of Bradyrhizobium japonicum to bind soybean lectin. The lectin-binding ability of bacteria grown on nitrocellulose filters was determined using peroxidase-labeled soybean lectin. The assay produced clear differences between strains known to be unable to bind soybean lectin and those which can. The assay gave results identical to those of the fluorescein isothiocyanate-soybean lectin-binding assay of T. V. Bhuvaneswari, S. G. Pueppke, and W. D. Bauer (1977, Plant Physiol. 60, 486-491) with regard both to the ability of particular B. japonicum strains to bind lectin and to the inhibition caused by N-acetyl-D-galactosamine. The method was used to screen Tn5-induced mutants of B. japonicum 2143 for their inability to bind soybean lectin. The procedure provides a sensitive and convenient method to screen Bradyrhizobium strains for the ability to bind soybean lectin.     

Versuche über Saisondimorphismus und verwandte Erscheinungen bei Ackerunkräutern

Ohne Zusammenfassung     

High Abundance of Neotropical Drosophilids (Diptera: Drosophilidae) in Four Cultivated Areas of Central Brazil

The drosophilid assemblages of four cultivated areas (soy, bean, corn, and orange plantations) grown in the core of the Neotropical region were analyzed by comparing their abundances and compositions. The collections, which were gathered using 38 banana traps, captured 12,560 drosophilids, including nine Neotropical and six exotic species. Most of the flies were collected in the bean (43%) and soy (42%) fields. The composition and relative abundance of species also varied among cultivated areas, with orange orchards presenting the highest relative abundance of exotics due to the dominance of the Afrotropical Zaprionus indianus (Gupta). Crop plantations were dominated by a Neotropical species, Drosophila cardini (Sturtevant), which has been shown to be well adapted to dry and disturbed environments. We discuss the drosophilid assemblages of the cultivated areas, comparing them with assemblages from neighbor urban and natural environments. The low drosophilid richness found in this study is similar to the richness found in urban environments and lower than the drosophilid richness of forests, supporting a pattern already known for other taxa. The high abundance of drosophilids in cultivated areas, as well as the dominance of a Neotropical species (D. cardini) in the crop assemblages, was a surprising result.     

Inhibition of FLT3 and PDGFR tyrosine kinase activity by bis(benzo[b]furan-2-yl)methanones

A series of bis(benzo[b]furan-2-yl)methanones was synthesized and tested for inhibition of FLT3 and PDGFR autophosphorylation. Mostly, C-5 substitution leads to PDGFR selectivity, which was strongest in the case of the 5,5'-dimethoxy derivative. The 5,5'-diamino and the 6,6'-dihydroxy compounds are more active at FLT3. At both kinases, the potency of the best inhibitors approaches IC50 values of ca. 0.5 microM. Molecular modeling studies suggest that the bisbenzofuranylmethanones are able to fit into the same binding site as their indolyl analogues which have been suggested to form a bidentate hydrogen bridge with the backbone in the hinge regions. The loss of one H bond by the NH-O exchange might be partially compensated by, for example, the weak interaction of one furanyl oxygen with FLT3 Cys-828.     

Loss of heterozygosity atBRCA1/2 loci in hereditary and sporadic ovarian cancers

Loss of heterozygosity atBRCA1/2 loci in breast and ovarian tumors is a suggested risk factor for germlineBRCA1/2 mutation status. We evaluated the presence of losses of selected microsatellite markers localized on chromosomes 17 and 13q in hereditary and sporadic ovarian tumors. 151 consecutive primary ovarian tumors (including 21 withBRCA1/2 mutations and 130 without the mutations) were screened for loss of heterozygosity at loci on chromosomes 17 and 13q. Losses of heterozygosity of at least one microsatellite marker localized on chromosomes 17 and 13q were revealed in 123 (81.5%) and 104 (68.9%) tumors, respectively. Losses of all informative markers on chromosomes 17 and 13 occurred in 30 (19.9%) and 31 (20.5%) tumors, respectively. There was no difference in the frequency of losses atBRCA1 intragenic markers (D17S855 and D17S1323) between BRCA1-positive and BRCA1-negative patients. The frequency of losses on chromosome 17 was higher in high-grade than in low-grade carcinomas. Loss of heterozygosity on chromosomes 17 and 13q is a frequent phenomenon in both hereditary and sporadic ovarian cancers. The frequency of losses atBRCA1 intragenic markers in the ovarian tumor tissue is not strongly related to the presence ofBRCA1 germline mutations.     

NinaB Is Essential for Drosophila Vision but Induces Retinal Degeneration in Opsin-deficient Photoreceptors

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase β-carotenoid-15,15′-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules

Soybean (Glycine max) nodules formed by inoculation with either an effective strain or an ineffective (noninvasive, nodule-forming) strain of Bradyrhizobium japonicum were assayed for changes in developmental patterns of carbon metabolic enzymes of the plant nodule cells. Of the enzyme activities measured, only sucrose synthase, glutamine synthetase, and alcohol dehydrogenase were altered in the ineffective nodules relative to the effective nodules. Sucrose synthase and glutamine synthetase activities were greatly reduced, whereas alcohol dehydrogenase activity was elevated. Dark-induced senescence severely affected sucrose synthase but had little, if any, effect on the other enzymes measured. The developmental patterns of the anaerobically induced enzymes, aldolase and alcohol dehydrogenase, were different from those expected, implying that their development is not regulated solely by oxygen deprivation. However, anaerobic treatment of nodules resulted in responses similar to those enzymes in maize. The developmental profiles of the carbon metabolic enzymes suggest that carbohydrates are metabolized via the sucrose synthase and pentose phosphate pathways. This route of carbon metabolism, compared to glycolysis, would reduce the requirement of ATP for carbohydrate catabolism, generate NADPH for biosynthetic reactions, and provide intermediates for plant secondary metabolism.