Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

Expression Vectors for the Rapid Purification of Recombinant Proteins in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We describe the construction of six novel plasmid-based IPTG-inducible expression vectors for Bacillus subtilis and related species. While one vector allows intracellular production of recombinant proteins, the second provides a strong secretion signal. The third vector allows addition of the c-Myc epitope tag, and the remaining three vectors provide the purification tags His and Strep. The versatility of all six vectors was demonstrated by the insertion of several reporter genes and by their regulated overexpression. Recombinant proteins with a His- or Strep-tag could be purified to near homogeneity in a single step.     

Dynamic changes in nitric oxide and mitochondrial oxidative stress with site-dependent differential tissue response during anoxic preconditioning in rat heart

In this study, dynamic changes in nitric oxide (NO) and mitochondrial superoxide (O(2)(*-)) were examined during anoxic preconditioning (AP) in rat heart model. AP and anoxia-reoxygenation (A/R) were performed on isolated hearts and single cardiomyocytes. The cellular insult in the form of infarct size and DNA damage were localized and correlated with NO synthases (endothelial and inducible) expression levels. The results showed that endocardium was the most affected region in AP groups, whereas the larger area of infarct was confined to mid- and epicardium in the A/R group. Interestingly, a high-level expression of immunofluorescent NO synthases was restricted to viable areas in the AP. In contrast to the gradual increase in O(2)(*-) level that occurred in the AP group, a sudden massive increase in its level was demonstrated at the onset of reoxygenation in the A/R group. The observed increase in NO production during reoxygenation in the AP group was attenuated by inducible NO synthase inhibitor. The study revealed, on a real-time basis, the role played by preconditioning for modulating NO and O(2)(*-) levels on behalf of cell survival. The results afford a better understanding of cardiac-adapting mechanism during AP and the role of inducible NO synthase in this important phenomenon.     

Partitioning and inactivation of viruses by the caprylic acid precipitation followed by a terminal pasteurization in the manufacturing process of horse immunoglobulins

Caprylic acid (octanoic acid), has been used for over 50 years as a stabilizer of human albumin during pasteurization. In addition caprylic acid is of great interest, by providing the advantage of purifying mammalian immunoglobulins and clearing viruses infectivity in a single step. Exploiting these two properties, we sequentially used the caprylic acid precipitation and the pasteurization to purify horse hyperimmune globulins used in the manufacturing of Sérocytol. To evaluate the effectiveness of the process for the removal/inactivation of viruses, spiking studies were carried out for each dedicated step. Bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), encephalomyocarditis virus (EMCV) and minute virus of mice (MVM) were used for the virological validation. Our data show that the treatment with caprylic acid 5% (v/v) can effectively be used as well to purify or to ensure viral safety of immunoglobulins. Caprylic acid precipitation was very efficient in removing and/or inactivating enveloped viruses (PRV, BVDV) and moderately efficient against non-enveloped viruses (MVM, ECMV). However the combination with the pasteurization ensured an efficient protection against both enveloped and non-enveloped viruses. So that viruses surviving to the caprylic acid precipitation will be neutralized by pasteurization. Significant log reduction were achieved > or =9 log(10) for enveloped viruses and 4 log(10) for non-enveloped viruses, providing the evidence of a margin of viral safety achieved by our manufacturing process. Its a simple and non-expensive manufacturing process of immunoglobulins easily validated that we have adapted to a large production scale with a programmable operating system.     

Phylogeographic structure and late Quaternary population history of the Japanese oak Quercus mongolica var. crispula and related species revealed by chloroplast DNA variation

Generally, oaks dominate the broadleaf deciduous forests in Japan. The genetic variation in 6 cpDNA regions (trnT-trnL, trnL-trnF, atpB-rbcL, and trnH-psbA speacers, trnL intron, and matK gene) with regard to the Japanese oak (Quercus mongolica var. crispula) and 3 related species in the section Prinus (Q. serrata, Q. dentata and Q. aliena) was investigated in 598 trees belonging to 44 populations distributed throughout the Japanese archipelago. Additional samples were collected from Korea, China, and Russia (Sakhalin). Thirteen haplotypes (I to XIII) were identified on the bases of 15 nucleotide substitutions and 3 indels. Haplotypes I and II were discovered in northeastern Japan, whereas haplotypes III to IX were distributed in southwestern Japan. The boundary distinguishing these 2 groups was located in central Japan coincident with the Itoigawa-Shzuoka tectonic line. Haplotype I was also found in Sakhalin, whereas haplotypes VI, VII, VIII, X, XI, XII, and XIII were found in Korea and China. Four oak species in the same location shared identical haplotypes, suggesting cpDNA introgression by occasional hybridization. Both the values of total haplotype diversity (HT) and haplotype diversity within populations (HS) in Q. mongolica var. crispula were higher in the southwestern populations than in the northeastern populations. A haplotype network indicated that haplotype VI is the ancestral haplotype. The presence of identical haplotypes in Korea, China, and Japan suggested that the haplotypes diversified on the Eurasian continent before the last glacial period. The difference in genetic structure between the northeastern and southwestern regions indicates a difference in the history of migration and recolonization in Japan during the last glacial period.     

A novel lepidopteran sex pheromone produced by females of a Lithosiinae species, Lyclene dharma dharma, in the family of Arctiidae

Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (I-III), which strongly stimulated male antennae. Using GC-MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.     

Selenoproteins of the thyroid gland: expression, localization and possible function of glutathione peroxidase 3

The thyroid gland has an exceptionally high selenium content, even during selenium deficiency. At least 11 selenoproteins are expressed, which may be involved in the protection of the gland against the high amounts of H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern blotting experiments, glutathione peroxidases (GPx) 1 and 4 and selenoprotein P were moderately expressed, occurring selectively in the follicular cells and in leukocytes of germinal follicles of thyroids affected by Hashimoto's thyroiditis. Selenoprotein 15 was only marginally expressed and distributed over all cell types. GPx3 mRNA was exclusively localized to the thyrocytes, showed the highest expression levels and was down-regulated in 5 of 6 thyroid cancer samples as compared to matched normal controls. GPx3 could be extracted from thyroidal colloid by incubation with 0.5% sodium dodecyl sulfate indicating that this enzyme is (i) secreted into the follicular lumen and (ii) loosely attached to the colloidal thyroglobulin. These findings are consistent with a role of selenoproteins in the protection of the thyroid from possible damage by H2O2. Particularly, GPx3 might use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form present in the colloid.     

Whole genome analysis of Vietnamese G2P[4] rotavirus strains possessing the NSP2 gene sharing an ancestral sequence with Chinese sheep and goat rotavirus strains

Because imminent introduction into Vietnam of a vaccine against Rotavirus A is anticipated, baseline information on the whole genome of representative strains is needed to understand changes in circulating strains that may occur after vaccine introduction. In this study, the whole genomes of two G2P[4] strains detected in Nha Trang, Vietnam in 2008 were sequenced, this being the last period during which virtually no rotavirus vaccine was used in this country. The two strains were found to be > 99.9% identical in sequence and had a typical DS‐1 like G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2 genotype constellation. Analysis of the Vietnamese strains with > 184 G2P[4] strains retrieved from GenBank/EMBL/DDBJ DNA databases placed the Vietnamese strains in one of the lineages commonly found among contemporary strains, with the exception of the NSP2 and NSP4 genes. The NSP2 genes were found to belong to a previously undescribed lineage that diverged from Chinese sheep and goat rotavirus strains, including a Chinese rotavirus vaccine strain LLR with 95% nucleotide identity; the time of their most recent common ancestor was 1975. The NSP4 genes were found to belong, together with Thai and USA strains, to an emergent lineage (VIII), adding further diversity to ever diversifying NSP4 lineages. Thus, there is a need to enhance surveillance of locally‐circulating strains from both children and animals at the whole genome level to address the effect of rotavirus vaccines on changing strain distribution.