<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of economically important oilseed rape cultivars

Oilseed rape is considered relatively recalcitrant for genetic modification. This work was performed to establish conditions for effective transformation and regeneration of commercially used cultivars Campino, Haydn, Heros, Hunter and Topas (Brassica napus L.). Cotyledonary petioles and hypocotyls (obtained from the seedlings grown under dark conditions) as a source of explants were used. Our experiments revealed that a lower selection pressure in combination with postponing of selection by 14 days after co-cultivation resulted in recovery of transgenic plants from all cultivars. Transformants were obtained with efficiency from 1 to 5.5%. Histochemically GUS-positive plants were analysed by PCR using primers corresponding to the internal fragments of gus and nptII genes. The transgenic nature was confirmed by Southern blot analyses using a specific nptII probe. This work enriches the list of oilseed rape cultivars available for genetic modification.     

Immunization of Chlamydia pneumoniae (Cpn)-Infected Apobtm2SgyLdlrtm1Her/J Mice with a Combined Peptide of Cpn Significantly Reduces Atherosclerotic Lesion Development

Objective

To investigate the antigenic effect of a peptide containing two epitopes of Chlamydia pneumoniae (Cpn) on atherosclerotic lesion formation in mice infected with Cpn.

Materials and Methods

Six-week-old Apob^tm2SgyLdlr^tm1Her/J mice were immunized using a repetitive immunization multiple-sites strategy with KLH-conjugated peptides derived from the major outer membrane protein and the putative outer membrane protein 5 of Cpn. Mice were fed a high-fat diet and infected with Cpn twice during the 10-week diet period. Lesions were evaluated histologically; local and systemic immune responses were analyzed by immunohistochemistry of aorta samples and cytokine measurements in plasma samples and splenocyte supernatants.

Results

Mice immunized with the combined Cpn peptide showed a greater reduction in lesion size compared to mice immunized with either epitope alone [54.7% vs 39.8% or 41.72%] and was also associated with a significant decrease in lesion area in descending aortas compared with those in controls (88.9% for combined Cpn peptide, 81.9% for MOMP peptide and 75.7% for Omp5, respectively). This effect was associated with a shift in the cellular composition of plaques towards decreased inflammatory cell and increased regulatory T-cell content. Additionally, the effect was also connected with decreased secretion of proinflammatory cytokines and increased production of anti-inflammatory cytokines demonstrated in plasma and in supernatant on stimulated spleen cells.

Conclusions

Atherosclerotic lesion formation may be promoted by Cpn infection in the presence of a high-fat diet, and reduced by immunization with the combined Cpn peptide. The combined peptide has more potential than either epitope alone in reducing atherosclerotic lesion development through Treg expansion.     

Cell volume and the regulation of apoptotic cell death

Apoptosis is a physiological mechanism allowing for the removal of abundant or potentially harmful cells. The hallmarks of apoptosis include degradation of cellular DNA, exposure of phosphatidylserine at the outer leaflet of the cell membrane and cell shrinkage. Phosphatidylserine exposure favours adhesion to macrophages with subsequent phagocytosis of the shrunken apoptotic particles. The interaction of cell volume regulatory mechanisms and apoptosis is illustrated in two different model systems, i.e. (a) lymphocyte apoptosis following stimulation of CD95 receptor and (b) erythrocyte apoptosis upon cell shrinkage. (a) Triggering of CD95 in Jurkat T lymphocytes is paralleled by activation of cell volume regulatory Cl- channels, inhibition of the Na+/H+ exchanger and osmolyte release. The latter coincides with cell shrinkage, DNA fragmentation and phosphatidylserine exposure. CD95 stimulation leads to early inhibition of the voltage gated K+ channel Kv1.3, which may contribute to the inhibition of the Ca2+ release activated Ca2+ channel I(CRAC). (b) Osmotic shock of erythrocytes activates a cell volume regulatory cation conductance allowing the entry not only of Na+ but of Ca2+ as well. Increased cytosolic Ca2+ stimulates a scramblase which disrupts the phosphatidylserine asymmetry of the cell membrane, leading to phosphatidylserine exposure. The cation conductance is further activated by oxidative stress and energy depletion and inhibited by Cl-. Shrinkage of erythrocytes stimulates in addition a sphingomyelinase with subsequent formation of ceramide which potentiates the effect of cytosolic Ca2+ on phosphatidylserine. In conclusion, cell volume-sensitive mechanisms participate in the triggering of apoptosis following receptor stimulation or cell injury.     

Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression

Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.     

Chitosomes lack concanavalin-A-binding sites

Whether intact or dissociated with digitonin, chitosomes isolated from the fungusMucor rouxii lack the ability to bind concanavalin A. The absence of external or internal concanavalin A-binding sites distinguishes the chitosome membrane no only from plasma membrane but also from membranes of other organelles (endoplasmic reticulum, mitochondrion, vacuole). This differential binding ability was used to partially separate chitosomal chitin synthetase from major membranes in a crude cell-free extract ofM. rouxii.     

Fine‐tuned regulation of the K+/H+ antiporter KEA3 is required to optimize photosynthesis during induction

KEA3 is a thylakoid membrane localized K⁺/H⁺ antiporter that regulates photosynthesis by modulating two components of proton motive force (pmf), the proton gradient (?pH) and the electric potential (?ψ). We identified a mutant allele of KEA3, disturbed proton gradient regulation (dpgr) based on its reduced non‐photochemical quenching (NPQ) in artificial (CO₂‐free with low O₂) air. This phenotype was enhanced in the mutant backgrounds of PSI cyclic electron transport (pgr5 and crr2‐1). In ambient air, reduced NPQ was observed during induction of photosynthesis in dpgr, the phenotype that was enhanced after overnight dark adaptation. In contrast, the knockout allele of kea3‐1 exhibited a high‐NPQ phenotype during steady state in ambient air. Consistent with this kea3‐1 phenotype in ambient air, the membrane topology of KEA3 indicated a proton efflux from the thylakoid lumen to the stroma. The dpgr heterozygotes showed a semidominant and dominant phenotype in artificial and ambient air, respectively. In dpgr, the protein level of KEA3 was unaffected but the downregulation of its activity was probably disturbed. Our findings suggest that fine regulation of KEA3 activity is necessary for optimizing photosynthesis.     

MSMEG_4626 ribonuclease from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The MSMEG_4626 gene was cloned from Mycobacterium smegmatis MC2 155. It codes for a protein of 1,037 amino acids, identified as ribonuclease E by matching to the protein family HMM TIGR00757. The protein was expressed and purified. Although its calculated molecular weight is 112.7 kDa, it has an aberrant mobility in SDS-polyacrylamide gels, like other ribonuclease E enzymes (it migrates as a 180 kDa protein). The central part of the protein displays high similarity to the catalytic domains of other RNase E enzymes. Mass spectrometric analysis revealed the presence of the chaperonin GroEL, ribosomal proteins, a negative regulator of genetic competence and GTP pyrophosphokinase in the affinity-purified preparation. It is a very unstable protein; despite the use of protease inhibitors in addition to the full-length RNase E its proteolytic fragments were detected.