Contribution of Glucosinolate Transport to Arabidopsis Defense Responses

Accumulation of glucosinolates, a class of defense-related secondary metabolites found almost exclusively in the Capparales, is induced in response to a variety of biological stresses. It is often assumed that elevated glucosinolate levels result from de novo biosynthesis, but glucosinolate transport from other parts of the plant to the site of herbivory or pathogen infection can also contribute to the defense response. Several studies with Arabidopsis and other crucifers have demonstrated that glucosinolates from vegetative tissue are transported to developing seeds. Here we discuss evidence that long-chain aliphatic glucosinolates are transported to the site of herbivory in response to Myzus persicae (green peach aphid) feeding on Arabidopsis.Key Words: glucosinolate, transport, graft, Arabidopsis, Myzus persicae, aphid     

Effects of the number of markers per haplotype and clustering of haplotypes on the accuracy of QTL mapping and prediction of genomic breeding values

The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r² value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis>

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.     

Delivery of antibodies to the cytosol: Debunking the myths

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG₁ Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.     

Rapid estimation of numbers of whiteflies (Hemiptera: Aleurodidae) and thrips (Thysanoptera: Thripidae) on sticky traps

The presence or absence of greenhouse whiteflies, Trialeurodes vaporariorum Westwood, and thrips, primarily western flower thrips, Frankliniella occidentalis (Pergande), in cells of a grid laid over 7.6 cm by 12.7 cm sticky traps was used to estimate the population density of these pests on the trap. The method accurately predicted trap population densities of between 15 and 192 individuals per side for thrips on blue and yellow traps and between 15 and 168 whiteflies per side on yellow traps. The distribution of both whiteflies and thrips tended to be clustered on the sides and upper edge of the traps. The method is useful in giving a far more rapid estimate than counting individuals, particularly at high population densities.     

Compartmentalization and actin binding properties of ABP-50: the elongation factor-1 alpha of Dictyostelium.

ABP-50 is the elongation factor-1 alpha (EF-1 alpha) of Dictyostelium discoideum (Yang et al.: Nature 347:494-496, 1990). ABP-50 is also an actin filament binding and bundling protein (Demma et al.: J. Biol. Chem. 265:2286-2291, 1990). In the present study we have investigated the compartmentalization of ABP-50 in both resting and stimulated cells. Immunofluorescence microscopy shows that in addition to being colocalized with F-actin in surface extensions in unstimulated cells, ABP-50 exhibits a diffuse distribution throughout the cytosol. Upon addition of cAMP, a chemoattractant, ABP-50 becomes localized in the filopodia that are extended as a response to stimulation. Quantification of ABP-50 in Triton-insoluble and -soluble fractions of resting cells indicates that 10% of the total ABP-50 is recovered in the Triton cytoskeleton, while the remainder is in the soluble cytosolic fraction. Stimulation with cAMP increases the incorporation of ABP-50 into the Triton cytoskeleton. The peak of incorporation of ABP-50 at 90 sec is concomitant with filopod extension. Immunoprecipitation of the cytosolic ABP-50 from unstimulated cells using affinity-purified polyclonal anti ABP-50 results in the coprecipitation of non-filamentous actin with ABP-50. Purified ABP-50 binds to G-actin with a Kd of approximately 0.09 microM. The interaction between ABP-50 and G-actin is inhibited by GTP but not by GDP, while the bundling of F-actin by ABP-50 is unaffected by guanine nucleotides. We conclude that a significant amount of ABP-50 is bound to either G- or F-actin in vivo and that the interaction between ABP-50 and F-actin in the cytoskeleton is regulated by chemotactic stimulation.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

Isolation of an abundant 50,000-dalton actin filament bundling protein from Dictyostelium amoebae

A monomeric actin bundling protein with a native molecular weight of approximately 50,000 (ABP-50) has been isolated from amoebae of Dictyostelium discoideum. ABP-50 cross-links F-actin to form tightly packed bundles, some of which are highly ordered. It exhibits a Kd of 2.1 microM and a molar ratio to actin of 1:1 in bundles. Calcium and ATP at physiological concentrations have no effect on these activities. ABP-50 is immunologically unrelated to 30-kDa protein, a previously described bundling protein from Dictyostelium. Immunofluorescence with affinity-purified polyclonal antibodies indicates that ABP-50 is localized in regions of the amoeboid cell cortex containing actin bundles. The molar ratio of ABP-50 to actin is approximately 1:5 in vivo. Therefore, the abundance of ABP-50 suggests that it may be responsible for the majority of the bundling activity in these cells.     

Relaxin-3 and RXFP3 expression, and steroidogenic actions in the ovary of teleost fish

Relaxins are peptides similar in secondary structure to insulins. In teleost genomes, five or six relaxin genes have been identified. Two relaxins group closely with mammalian relaxin-3 on phylogenetic analysis and are named relaxin-3a and b. We refer to the remainder as relaxins c to f. Ovarian expression of relaxin-3a, d and f genes, and the relaxin-3 receptor gene Rxfp3, was studied in Danio rerio using RT-PCR. Immunohistochemistry was used to determine the distribution of relaxin-3 peptides and RXFP3 in the ovary of Fundulus heteroclitus (killifish). Thirdly, enzyme immunoassays and ovarian follicular culture were used to determine the effect of treatment with human recombinant relaxin-3 on the production of 17beta-estradiol and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in killifish ovarian follicles. Relaxin-3a, d, f, and Rxfp3 genes were expressed regardless of sex or reproductive condition. Relaxin-3 immunostaining was present in mid to late follicular stages within cortical alveoli of the oocyte cytopasm, whereas receptor staining was localized to follicular cells. Treatment with relaxin-3 enhanced 17beta-estradiol production in early and late maturing follicles, but did not have an effect in vitellogenic follicles. Relaxin-3 appeared to suppress the release of MIS production. This suggests that relaxin peptides may be involved with estradiol-dependant events in follicular development.