Nucleotide and nucleoside involvement in immunomodulation in experimental Chagas disease

Whether the Arg913Gln variation (rs11643718, G/A) of SLC12A3 contributes to diabetic nephropathy (DN) remains controversial. We undertook a case–control study to evaluate the association of the SLC12A3-Arg913Gln variation with the risk of end-stage renal disease (ESRD) in Chinese type 2 diabetes mellitus (T2DM) patients undergoing hemodialysis, and analyzed the genotype–phenotype interaction. Unrelated Chinese T2DM patients (n = 372) with diabetic retinopathy were classified into the non-DN (control) group (n = 151; duration of T2DM >15 years, no signs of renal involvement) and the DN–ESRD group (n = 221; ESRD due to T2DM, receiving hemodialysis). Polymerase chain reaction-direct sequencing was used to genotype the SLC12A3-Arg913Gln variation for all participants. The frequency of the GA+AA genotype in the DN–ESRD group was significantly higher than that of the non-DN group (23.1 vs. 9.9%; adjusted OR 2.2 (95% CI 1.3–4.5), P = 0.019). In the non-DN group, GA+AA carriers had a significantly higher urinary albumin excretion rate (UAER) and diastolic blood pressure compared with GG carriers (both P < 0.05). The SLC12A3-Arg913Gln variation may be associated with increased blood pressure and UAER and, therefore, could be used to predict the development and progression of DN–ESRD in Chinese T2DM patients undergoing hemodialysis.     

Genetic loci controlling lethal cell death in tomato caused by viral satellite RNA infection

Cucumber mosaic virus (CMV) associated with D satellite RNA (satRNA) causes lethal systemic necrosis (LSN) in tomato (Solanum lycopersicum), which involves programmed cell death. No resistance to this disease has been found in tomato. We obtained a line of wild tomato, S. habrochaitis, with a homogeneous non-lethal response (NLR) to the infection. This line of S. habrochaitis was crossed with tomato to generate F1 plants that survived the infection with NLR, indicating that NLR is a dominant trait. The NLR trait was successfully passed on to the next generation. The phenotype and genotype segregation was analyzed in the first backcross population. The analyses indicate that the NLR trait is determined by quantitative trait loci (QTL). Major QTL associated with the NLR trait were mapped to chromosomes 5 and 12. Results from Northern blot and in situ hybridization analyses revealed that the F1 and S. habrochaitis plants accumulated minus-strand satRNA more slowly than tomato, and fewer vascular cells were infected. In addition, D satRNA-induced LSN in tomato is correlated with higher accumulation of the minus-strand satRNA compared with the accumulation of the minus strand of a non-necrogenic mutant D satRNA.     

On-line identification of further flavone C- and O-glycosides from sugarcane (Saccharum officinarum L., Gramineae) by HPLC-UV-MS

HPLC-UV-MS (APCI-MS/MS and CID/MS) was utilised for the identification of eight additional flavone glycosides from sugarcane (Saccharum officinarum L., Gramineae) extracts of bagasse, leaves and juice ('garapa'). Relevant information about substitution patterns was obtained through UV detection using post-column addition of shift reagents, while tandem MS provided structural information confirming the proposed structures of the C-glycosides vitexin, orientin, luteolin-8-C-(rhamnosylglucoside), 4',5'-dimethyl-luteolin-8-C-glycoside and the isomeric pair schaftoside-isoschaftoside, besides the O-glycosides tricin-7-O-neohesperidoside and tricin-7-O-glycoside.     

Exploring substrate binding in homoprotocatechuate 2,3-dioxygenase using isothermal titration calorimetry

Homoprotocatechuate 2,3-dioxygenase (HPCD) is a member of the extradiol dioxygenase family of non-heme iron enzymes. These enzymes catalyze the ring-cleavage step in the aromatic degradation pathway commonly found in soil bacteria. In this study, isothermal titration calorimetry (ITC) is used to measure the equilibrium constant (K?=?1.1?±?0.6?×?10(6)) and enthalpy change (ΔH?=?-17.0?±?1.7?kcal/mol) associated with homoprotocatechuate binding to HPCD. The ITC data are consistent with the release of approximately 2.6 protons upon binding of the substrate to HPCD. These results raise new questions regarding the relationships between substrate, protein, and the oxygen activation mechanism for this class of non-heme metalloenzymes.     

Characterization of complexes formed between TSG-6 and inter-alpha-inhibitor that act as intermediates in the covalent transfer of heavy chains onto hyaluronan

The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.     

Seasonal and diel transitions in physiology and behavior in the migratory dark-eyed junco

Body mass, fat stores, activities of lipogenic and lipolytic enzymes, and plasma corticosterone were measured throughout seasonal and diel transitions from fall through spring encompassing the non-migratory stages of early and mid winter, the prealternate molt, and the spring migratory stage in captive dark-eyed juncos to determine the physiological mechanisms underlying adaptations for migration. On a seasonal basis, lipid enzymes and corticosterone varied little throughout the stages even though the birds underwent dramatic alterations in mass, fat deposition, behavior, and activation of the reproductive axis. By contrast, diel changes were found in lipogenesis, lipolysis, muscle lipoprotein lipase, and plasma corticosterone when comparing birds in the two phases of spring migration--active flight and resting, as during times of stopover. In these two phases of migration, coordination of the lipogenic and lipolytic systems appear to maximize storage of fatty acids during rest and delivery/utilization during flight. Diel patterns of corticosterone revealed fairly consistent peaks during the night time (23:00) throughout the nonmigratory period. The profile of this pattern altered during the migratory period with variation between the flight and resting phases. In sum, the results from these captive studies offer a new approach for studying the regulation of migratory physiology in free-living birds.     

Ecological correlates of anuran exercise physiology

Summary Studies of exercise physiology of anuran amphibians have led to the suggestion that there is a dichotomy between species that depend upon movement to escape from predators and species that utilize static defenses. This generalization has been based upon a limited taxonomic survey and it contrasts with morphological, ecological, and behavioral studies that have revealed diverse and complex interrelationships among these features of anuran biology. We tested the hypothesis of a dichotomy of physiological types among anurans by measuring aerobic and anaerobic metabolism during maximum exercise for 17 species representing seven families and a variety of ecological types and locomotor modes. All degrees of dependence upon aerobic and anaerobic power input were found among the 17 species and the variation did not follow phylogenetic divisions. No single, simple prediction of the predominant source of power utilized for activity by the anurans we studied is possible. Predator avoidance behavior was not significantly correlated with the metabolic pattern. Predatory mode (active versus passive searchers) and mode of locomotion (non-jumpers versus jumpers) were correlated with dependence upon aerobic energy production and with each other. Reproductive behavior is probably another associated factor. The diversity of modes of power input among anurans is great and is intimately linked with numerous features of a species' biology. Single-factor explanations of this physiological characteristic are not appropriate.     

A dominant mutation in β-AMYLASE1 disrupts nighttime control of starch degradation in Arabidopsis leaves

Arabidopsis (Arabidopsis thaliana) leaves possess a mechanism that couples the rate of nighttime starch degradation to the anticipated time of dawn, thus preventing premature exhaustion of starch and nighttime starvation. To shed light on the mechanism, we screened a mutagenized population of a starvation reporter line and isolated a mutant that starved prior to dawn. The mutant had accelerated starch degradation, and the rate was not adjusted to time of dawn. The mutation responsible led to a single amino acid change (S132N) in the starch degradation enzyme BETA-AMYLASE1 (BAM1; mutant allele named bam1-2D), resulting in a dominant, gain-of-function phenotype. Complete loss of BAM1 (in bam1-1) did not affect rates of starch degradation, while expression of BAM1(S132N) in bam1-1 recapitulated the accelerated starch degradation phenotype of bam1-2D. In vitro analysis of recombinant BAM1 and BAM1(S132N) proteins revealed no differences in kinetic or stability properties, but in leaf extracts, BAM1(S132N) apparently had a higher affinity than BAM1 for an established binding partner required for normal rates of starch degradation, LIKE SEX FOUR1 (LSF1). Genetic approaches showed that BAM1(S132N) itself is likely responsible for accelerated starch degradation in bam1-2D and that this activity requires LSF1. Analysis of plants expressing BAM1 with alanine or aspartate rather than serine at position 132 indicated that the gain-of-function phenotype is not related to phosphorylation status at this position. Our results strengthen the view that control of starch degradation in wild-type plants involves dynamic physical interactions of degradative enzymes and related proteins with a central role for complexes containing LSF1.

A single amino acid change in Arabidopsis BETA-AMYLASE1 prevents coupling of night-time starch degradation to time of dawn and causes premature exhaustion of starch reserves in the dark.