Compensatory substitutions and the evolution of the mitochondrial 12S rRNA gene in mammals

12S ribosomal RNA (rRNA) gene sequences from a suite of mammalian taxa (13 placentals, 4 marsupials, 1 monotreme), for which phylogenetic relationships are well established based on independent criteria, were employed to study the evolution of this gene. Phylogenetic analysis of 12S sequences produces a phylogeny that agrees with expectations. Base composition provides evidence for directional symmetrical substitution pressure in loops; in stems, base composition is much more even. Rates of nucleotide substitution are lower in stems than loops. Patterns of nucleotide substitution show an overall preference for transitions over transversions, with this difference more profound in stems than loops. Among different transversion pathways, there is a wide range of transformation frequencies. An analysis of compensatory substitutions shows that there is strong evidence for their occurrence and that a weighting factor of 0.61 should be applied in phylogenetic analyses to account for the dependence of mutations at stem positions relative to positions where changes are independent. Among stem variables (i.e., stem length, interaction distance, substitution rates, G+C content, and the percentage of bases that are paired), several significant correlations were discovered, but stem length and interaction distance are uncorrelated with other variables.      

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.     

Ice algal assemblages and vertical export of organic matter from sea ice in the Barents Sea and Nansen Basin (Arctic Ocean)

Algal communities and export of organic matter from sea ice were studied in the offshore marginal ice zone (MIZ) of the northern Barents Sea and Nansen Basin of the Arctic Ocean north of Svalbard by means of ice cores and short-term deployed sediment traps. The observations cover a total of ten stations within the drifting pack ice, visited over a period of 3 years during the period of ice melt in May and July. Maximum flux of particulate organic carbon and chlorophyll a from the ice at 1 m depth (1,537 mg C m⁻² per day and 20 mg Chl a m⁻² per day) exceeded the flux at 30 m by a factor of 2 during spring, a pattern that was reversed later in the season. Although diatoms dominated the ice-associated algal biomass, flagellates at times revealed similarly high biomass and typically dominated the exported algal carbon. Importance of flagellates to the vertical flux increased as melting progressed, whereas diatoms made the highest contribution during the early melting stage. High export of ice-derived organic matter and phytoplankton took place simultaneously in the offshore MIZ, likely as a consequence of ice drift dynamics and the mosaic structure of ice-covered and open water characteristic of this region.     

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.     

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.