Thioredoxin targets in Arabidopsis roots

The importance of redox‐regulation in Arabidopsis thaliana roots has been investigated through the identification of the proteins interacting with thioredoxin (TRX), an ubiquitous thiol‐disulfide reductase. We have applied a proteomic approach based on affinity chromatography on a monocysteinic mutant of plastidial y‐type TRX used as a bait to trap putative partners in a crude extract of root proteins. Seventy‐two proteins have been identified, functioning mainly in metabolism, detoxification and response to stress, protein processing and signal transduction. This study allowed us to isolate 24 putative new targets and to propose the mevalonic acid‐dependent biosynthesis of isoprenoids as a new redox‐mediated process. The redox‐regulation of phenylpropanoid biosynthesis is also suggested, three enzymes of this pathway being retained on the column. We also provided experimental evidence that phenylammonia‐lyase was enzymatically more active when reduced by TRXy in root crude extract. Among the high number of partners involved in defense against stress we isolated from the column, we focused on plastidial monodehydroascorbate reductase and showed that its activity was dramatically increased in vitro in the presence of DTT‐reduced TRXy1 in root crude extracts. Our data strongly suggest that TRXy1 could be the physiological regulator of monodehydroascorbate reductase in root plastids.     

A genetic test for recruitment enhancement in Chesapeake Bay oysters, Crassostrea virginica, after population supplementation with a disease tolerant strain

Many of the methods currently employed to restore Chesapeake Bay populations of the eastern oyster, Crassostrea virginica, assume closed recruitment in certain sub-estuaries despite planktonic larval durations of 2–3 weeks. In addition, to combat parasitic disease, artificially selected disease tolerant oyster strains are being used for population supplementation. It has been impossible to fully evaluate these unconventional tactics because offspring from wild and selected broodstock are phenotypically indistinguishable. This study provides the first direct measurement of oyster recruitment enhancement by using genetic assignment tests to discriminate locally produced progeny of a selected oyster strain from progeny of wild parents. Artificially selected oysters (DEBY strain) were planted on a single reef in each of two Chesapeake Bay tributaries in 2002, but only in the Great Wicomico River (GWR) were they large enough to potentially reproduce the same year. Assignment tests based on eight microsatellite loci and mitochondrial DNA markers were applied to 1579 juvenile oysters collected throughout the GWR during the summer of 2002. Only one juvenile oyster was positively identified as an offspring of the 0.75 million DEBY oysters that were planted in the GWR, but 153 individuals (9.7%) had DEBY ×wild F1 multilocus genotypes. Because oyster recruitment was high across the region in 2002, the proportionately low enhancement measured in the GWR would not otherwise have been recognized. Possible causes for low enhancement success are discussed, each bearing on untested assumptions underlying the restoration methods, and all arguing for more intensive evaluation of each component of the restoration strategy.     

Comparative proteomic approaches for the isolation of proteins interacting with thioredoxin

Thioredoxin (TRX) is a small multifunctional protein with a disulfide active site involved in redox regulation. To gain insight into the numerous proteins able to interact with thioredoxin in Arabidopsis thaliana, we have compared three different proteomic procedures. In the two first approaches targets present in a mixture of soluble leaf proteins were reduced by the cytosolic TRX h3, then the new thiols were labeled either with radioactive iodoacetamide allowing specific detection (first method) or with a biotinylated thiol-specific compound allowing selective retention on an avidin column (second method). The third method involved a chromatography on a mutated TRX h3 column, which is able to covalently trap potential targets. All together, the three approaches enabled us to propose 73 proteins as being TRX-linked, and involved in various processes. Methods 1 and 3 were not only efficient with respectively 47 and 41 potential targets, but also complementary as only 26% of the targets were identified by both procedures. The second method with only 12 proteins was less efficient. However, this approach, as well as the first one when coupled with differential labeling of the cysteine residues, could be more informative about the cysteines involved in the thiol-disulfide interchange.     

Diabetes alters vascular mechanotransduction: pressure-induced regulation of mitogen activated protein kinases in the rat inferior vena cava

Background

Foam cell formation in diabetic patients often occurs in the presence of high insulin and glucose levels. To test whether hyperinsulinemic hyperglycemic conditions affect foam cell differentiation, we examined gene expression, cytokine production, and Akt phosphorylation in human monocyte-derived macrophages incubated with two types of oxidized low density lipoprotein (LDL), minimally modified LDL (mmLDL) and extensively oxidized LDL (OxLDL).

Methods and results

Using Affymetrix GeneChip^® arrays, we found that several genes directly related to insulin signaling were changed. The insulin receptor and glucose-6-phosphate dehydrogenase were upregulated by mmLDL and OxLDL, whereas insulin-induced gene 1 was significantly down-regulated. In hyperinsulinemic hyperglycemic conditions, modified LDL upregulated Akt phosphorylation and expression of the insulin-regulated aminopeptidase. The level of proinflammatory cytokines, IL-lβ, IL-12, and IL-6, and of a 5-lipoxygenase eicosanoid, 5-hydroxyeicosatetraenoic acid (5-HETE), was also increased.

Conclusion

These results suggest that the exposure of macrophages to modified low density lipoproteins in hyperglycemic hyperinsulinemic conditions affects insulin signaling and promotes the release of proinflammatory stimuli, such as cytokines and eicosanoids. These in turn may contribute to the development of insulin resistance.     

Evolutionary relationships among the Scarabaeini (Coleoptera: Scarabaeidae) based on combined molecular and morphological data

The Scarabaeini is an old world tribe of ball-rolling dung beetles that have origins dating back to at least the mid-upper Miocene (19-8 million years ago). The tribe has received little to no attention in morphological or molecular phylogenetics. We obtained sequence data from the mitochondrial cytochrome oxidase subunit I (1,197 bp) and 16S ribosomal RNA (461 bp) genes for 25 species of the Scarabaeini in an attempt to further resolve broad phylogenetic relationships within this tribe. Sequence data from both markers along with 216 morphological and 3 biological characters were analysed separately and combined. Independent analyses showed poorly resolved trees with many of the intermediate and basal nodes collapsed by low bootstrap values. Many sites in both genes exhibited strong A+T nucleotide bias and high interlineage divergences. The combined analysis revealed a number of well supported relationships such as the monophyly of the nocturnal species Scarabaeus satyrus, S. [Neateuchus] proboscideus, and S. zambesianus. Furthermore, the total evidence tree suggested to elevate S. (Pachysoma) to the status of an independent genus, Pachysoma, as a sister taxon to a clade containing Pachylomerus femoralis and Scarabaeus sensu lato. Within the latter, the following subgenera were maintained by the combination of data sets: S. (Scarabaeolus), S. (Sceliages), and S. (Kheper). Both, feeding specialisation and food relocation behaviour, were inferred to be polyphyletic in the Scarabaeini. Total evidence analysis found no support for common ancestry of Scarabaeini and Eucraniini.     

Bax-inhibiting peptide derived from mouse and rat Ku70

Bax is a proapoptotic protein that plays a key role in the induction of apoptosis. Ku70 has activities to repair DNA damage in the nucleus and to suppress apoptosis by inhibiting Bax in the cytosol. We previously designed peptides based on the amino acid sequence of Bax-binding domain of human Ku70, and showed that these peptides bind Bax and inhibit cell death in human cell lines. In the present report, we examined the biological activities of other pentapeptides, VPTLK and VPALR, derived from mouse and rat Ku70. Cells in culture accumulated FITC-labeled VPTLK and VPALR, indicating that these peptides are cell permeable (human, mouse, rat, and porcine cells were examined). These peptides bound to Bax and suppressed cell death in various cell types including primary cultured cells. These data suggest that such Bax inhibiting peptides from three mammalian species may be used to protect healthy cells from apoptotic injury under pathological conditions.     

Molecular dissection of a quantitative trait locus: a phenylalanine-to-tyrosine substitution in the transmembrane domain of the bovine growth hormone receptor is associated with a major effect on milk yield and composition

We herein report on our efforts to improve the mapping resolution of a QTL with major effect on milk yield and composition that was previously mapped to bovine chromosome 20. By using a denser chromosome 20 marker map and by exploiting linkage disequilibrium using two distinct approaches, we provide strong evidence that a chromosome segment including the gene coding for the growth hormone receptor accounts for at least part of the chromosome 20 QTL effect. By sequencing individuals with known QTL genotype, we identify an F to Y substitution in the transmembrane domain of the growth hormone receptor gene that is associated with a strong effect on milk yield and composition in the general population.     

Molecular identification and phylogeny of Armillaria isolates from South America and Indo-Malaysia

Armillaria root rot is a serious disease, chiefly of woody plants, caused by many species of Armillaria that occur in temperate, tropical and subtropical regions of the world. Very little is known about Armillaria in South America and Southeast Asia, although Armillaria root rot is well known in these areas. In this study, we consider previously unidentified isolates collected from trees with symptoms of Armillaria root rot in Chile, Indonesia and Malaysia. In addition, isolates from basidiocarps resembling A. novae-zelandiae and A. limonea, originating from Chile and Argentina, respectively, were included in this study because their true identity has been uncertain. All isolates in this study were compared, based on their similarity in ITS sequences with previously sequenced Armillaria species, and their phylogenetic relationship with species from the Southern Hemisphere was considered. ITS sequence data for Armillaria also were compared with those available at GenBank. Parsimony and distance analyses were conducted to determine the phylogenetic relationships between the unknown isolates and the species that showed high ITS sequence similarity. In addition, IGS-1 sequence data were obtained for some of the species to validate the trees obtained from the ITS data set. Results of this study showed that the ITS sequences of the isolates obtained from basidiocarps resembling A. novae-zelandiae are most similar to those for this species. ITS sequences for isolates from Indonesia and Malaysia had the highest similarity to A. novae-zelandiae but were phylogenetically separated from this species. Isolates from Chile, for which basidiocarps were not found, were similar in their ITS and IGS-1 sequences to the isolate from Argentina that resembled A. limonea. These isolates, however, had the highest ITS and IGS-1 sequence similarity to authentic isolates of A. luteobubalina and were phylogenetically more closely related to this species than to A. limonea.