Milk inhibits the biological activity of ricin

Ricin is a highly toxic protein produced by the castor plant Ricinus communis. The toxin is relatively easy to isolate and can be used as a biological weapon. There is great interest in identifying effective inhibitors for ricin. In this study, we demonstrated by three independent assays that a component of reconstituted powdered milk has a high binding affinity to ricin. We discovered that milk can competitively bind to and reduce the amount of toxin available to asialofetuin type II, which is used as a model to study the binding of ricin to galactose cell-surface receptors. Milk also removes ricin bound to the microtiter plate. In parallel experiments, we demonstrated by activity assay and by immuno-PCR that milk can bind competitively to 1 ng/ml ricin, reducing the amount of toxin uptake by the cells, and thus inhibit the biological activity of ricin. The inhibitory effect of milk on ricin activity in Vero cells was at the same level as by anti-ricin antibodies. We also found that (a) milk did not inhibit ricin at concentrations of 10 or 100 ng/ml; (b) autoclaving 10 and 100 ng/ml ricin in DMEM at 121 °C for 30 min completely abolished activity; and (c) milk did not affect the activity of another ribosome inactivating protein, Shiga toxin type 2 (Stx2), produced by pathogenic Escherichia coli O157:H7. Unlike ricin, which is internalized into the cells via a galactose-binding site, Stx2 is internalized through the cell surface receptor glycolipid globotriasylceramides Gb3 and Gb4. These observations suggest that ricin toxicity may possibly be reduced at room temperature by a widely consumed natural liquid food.     

Sulfite oxidase controls sulfur metabolism under SO2 exposure in Arabidopsis thaliana

In the present study, the significance of sulfite oxidase (SO) for sulfite detoxification and sulfur assimilation was investigated. In response to sulfur dioxide (SO(2)) exposure, a remarkable expansion of sulfate and a significant increase of GSH pool were observed in wild-type and SO-overexpressing Arabidopsis. These metabolic changes were connected with a negative feedback inhibition of adenosine 5'-phosphosulfate reductase (APR), but no alterations in gas exchange parameters or visible symptoms of injury. However, Arabidopsis SO-KO mutants were consistently negatively affected upon 600 nL L(-1) SO(2) exposure for 60 h and showed phenotypical symptoms of injury with small necrotic spots on the leaves. The mean g(H2O) was reduced by about 60% over the fumigation period, accompanied by a reduction of net CO(2) assimilation and SO(2) uptake of about 50 and 35%. Moreover, sulfur metabolism was completely distorted. Whereas sulfate pool was kept constant, thiol-levels strongly increased. This demonstrates that SO should be the only protagonist for back-oxidizing and detoxification of sulfite. Based on these results, it is suggested that co-regulation of SO and APR controls sulfate assimilation pathway and stabilizes sulfite distribution into organic sulfur compounds. In conclusion, a sulfate-sulfite cycle driven by APR and SO can be postulated for fine-tuning of sulfur distribution that is additionally used for sulfite detoxification, when plants are exposed to atmospheric SO(2).     

Assessment of Genetic Stability in Three Generations of In Vitro Propagated <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. Plantlets Using ISSR Markers

Tissue culture has been widely employed in Jatropha curcas L. for the clonal multiplication of superior genotypes. However, the evaluation of genetic stability is necessary to detect somaclonal variants. In this context, the present aim was to evaluate the genetic stability of J. curcas plantlets, obtained via indirect organogenesis, by means of ISSR markers. To supply the explant sources for in vitro propagation, the first generation of plants was produced from in vitro germination of J. curcas seeds. Fragments of cotyledonary leaves were inoculated into medium supplemented with 1.5 mg L^?1 BAP and 0.05 mg L^?1 of IBA for induction of callogenesis. The resulting calli were transferred to bud induction medium. Subsequently, the buds were cultured in medium for elongation, giving rise to the second generation of plants. These plants provided new buds, which were excised and subcultured in elongation medium, yielding a third generation of plants. To evaluate genetic stability in three plant generations, twelve ISSR primers were used, resulting in 124 bands showing 41.93 % of polymorphism. Increase was observed in the level of somaclonal variation (SV) over the generations. The present study reports, for the first time, the analysis of genetic stability in J. curcas plantlets regenerated via indirect organogenesis by means of ISSR markers. The results suggest that the indirect route is associated to higher levels of genetic instability, which also increased with successive subcultures. The ISSR markers were efficient in detecting SV, and the generated genetic variability may be useful for breeding programs.     

Oxidative Half-reaction of Arabidopsis thaliana Sulfite Oxidase: GENERATION OF SUPEROXIDE BY A PEROXISOMAL ENZYME*

Vertebrate forms of the molybdenum-containing enzyme sulfite oxidase possess a b-type cytochrome prosthetic group that accepts reducing equivalents from the molybdenum center and passes them on to cytochrome c. The plant form of the enzyme, on the other hand, lacks a prosthetic group other than its molybdenum center and utilizes molecular oxygen as the physiological oxidant. Hydrogen peroxide is the ultimate product of the reaction. Here, we present data demonstrating that superoxide is produced essentially quantitatively both in the course of the reaction of reduced enzyme with O₂ and during steady-state turnover and only subsequently decays (presumably noncatalytically) to form hydrogen peroxide. Rapid-reaction kinetic studies directly following the reoxidation of reduced enzyme demonstrate a linear dependence of the rate constant for the reaction on [O₂] with a second-order rate constant of k_ox = 8.7 × 10⁴ ± 0.5 × 10⁴ m⁻¹s⁻¹. When the reaction is carried out in the presence of cytochrome c to follow superoxide generation, biphasic time courses are observed, indicating that a first equivalent of superoxide is generated in the oxidation of the fully reduced Mo(IV) state of the enzyme to Mo(V), followed by a slower oxidation of the Mo(V) state to Mo(VI). The physiological implications of plant sulfite oxidase as a copious generator of superoxide are discussed.     

Conservation rules, their breakdown, and optimality in Caenorhabditis sinusoidal locomotion

Undulatory locomotion is common to nematodes as well as to limbless vertebrates, but its control is not understood in spite of the identification of hundred of genes involved in Caenorhabditis elegans locomotion. To reveal the mechanisms of nematode undulatory locomotion, we quantitatively analysed the movement of C. elegans with genetic perturbations to neurons, muscles, and skeleton (cuticle). We also compared locomotion of different Caenorhabditis species. We constructed a theoretical model that combines mechanics and biophysics, and that is constrained by the observations of propulsion and muscular velocities, as well as wavelength and amplitude of undulations. We find that normalized wavelength is a conserved quantity among wild-type C. elegans individuals, across mutants, and across different species. The velocity of forward propulsion scales linearly with the velocity of the muscular wave and the corresponding slope is also a conserved quantity and almost optimal; the exceptions are in some mutants affecting cuticle structure. In theoretical terms, the optimality of the slope is equivalent to the exact balance between muscular and visco-elastic body reaction bending moments. We find that the amplitude and frequency of undulations are inversely correlated and provide a theoretical explanation for this fact. These experimental results are valid both for young adults and for all larval stages of wild-type C. elegans. In particular, during development, the amplitude scales linearly with the wavelength, consistent with our theory. We also investigated the influence of substrate firmness on motion parameters, and found that it does not affect the above invariants. In general, our biomechanical model can explain the observed robustness of the mechanisms controlling nematode undulatory locomotion.     

Characterization of a new type of molybdenum cofactor-mutant in cell cultures of Nicotiana tabacum

Summary Four allelic putative cnx (molybdenum-cofactor defective) cell lines (O42, P12, P31 and P47) of Nicotiana tabacum var. Xanthi, biochemically and genetically distinct from N. tabacum var. Gatersleben cnxA mutants, were examined further. Their molybdenum-cofactor could efficiently reconstitute NADPH-nitrate reductase activity from Neurospora crassa mutant nit-1 extract only in the presence of exogenous molybdenum unlike that of the wild-type cofactor which could reconstitute NADPH-nitrate reductase activity in either the absence or presence of exogenous molybdenum. Loss of cofactor activity in vivo was not due to a defect in molybdenum uptake into the cells. In vitro nitrate reductase complementation between extracts of each of these four lines and a nia mutant showed that they possessed a functional nitrate reductase haemoflavoprotein subunit. Both constitutive molybdenum cofactor and NADH cytochrome c reductase activity were derepressed in the four cell lines. These results show that the four cell lines are indeed altered at a cnx locus, called cnxB, that the defect is probably in molybdenum processing and that there is a link between synthesis of functional molybdenum cofactor and nitrate reductase aporprotein.