Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

Improved method for transport of living cell cultures

An easy and cost-effective method for transport of living cell cultures which avoids the use of dry ice and prevents bacterial contamination is described. Cells are suspended in buffered culture medium in sealed and insulated 2 ml cryovials and are able to grow and survive in substantial numbers during several days of storage and shipment at ambient temperature. Replating results in an identical repopulation in all cell lines. Not only tumor cells but also fibroblasts seem to tolerate well this improved method for shipment.     

Disentangling multiple drivers of pollination in a landscape-scale experiment

Animal pollination is essential for the reproductive success of many wild and crop plants. Loss and isolation of (semi-)natural habitats in agricultural landscapes can cause declines of plants and pollinators and endanger pollination services. We investigated the independent effects of these drivers on pollination of young cherry trees in a landscape-scale experiment. We included (i) isolation of study trees from other cherry trees (up to 350 m), (ii) the amount of cherry trees in the landscape, (iii) the isolation from other woody habitats (up to 200 m) and (iv) the amount of woody habitats providing nesting and floral resources for pollinators. At the local scale, we considered effects of (v) cherry flower density and (vi) heterospecific flower density. Pollinators visited flowers more often in landscapes with high amount of woody habitat and at sites with lower isolation from the next cherry tree. Fruit set was reduced by isolation from the next cherry tree and by a high local density of heterospecific flowers but did not directly depend on pollinator visitation. These results reveal the importance of considering the plant''s need for conspecific pollen and its pollen competition with co-flowering species rather than focusing only on pollinators’ habitat requirements and flower visitation. It proved to be important to disentangle habitat isolation from habitat loss, local from landscape-scale effects, and direct effects of pollen availability on fruit set from indirect effects via pollinator visitation to understand the delivery of an agriculturally important ecosystem service.     

Enhanced biodegradation of hexachlorocyclohexane (HCH) in contaminated soils via inoculation with Sphingobium indicum B90A

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30°C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15–20% culturability after 30 days storage at 30°C, whereas full culturability was maintained when cells were stored frozen at −20°C. On the contrary, cells stored on corncob degraded γ-HCH faster than those that had been stored frozen, with between 15 and 85% of γ-HCH disappearance in microcosms within 20 h at 30°C. Soil microcosm tests at 25°C confirmed complete mineralization of [¹⁴C]-γ-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.     

Increased expression of costimulatory markers CD134 and CD80 on interleukin-17 producing T cells in patients with systemic lupus erythematosus

Introduction  

There is growing evidence that interleukin 17 (IL-17) producing T cells are involved in the pathogenesis of systemic lupus erythematosus (SLE). Previous studies showed that increased percentages of T-cell subsets expressing the costimulatory molecules CD80 and CD134 are associated with disease activity and renal involvement in SLE. The aim of this study was to investigate the distribution and phenotypical characteristics of IL-17 producing T-cells in SLE, in particular in patients with lupus nephritis, with emphasis on the expression of CD80 and CD134.     

Fluorescence immunolocalization of bound atrazine residues in plant tissue

Bound atrazine was detected inElodea canadensis by an improved immunohistochemical fluorescence procedure using anti-triazine antibodies from rabbits, biotin-labelled anti-rabbit immunoglobulin G and streptavidin-phycoerythrin conjugate. Whereas no labelling was found in control plants grown in charcoal-filtered, atrazine-free water, the labelling of plants obtained from their natural habitat and grown in tap water was sometimes nearly as high as in samples loaded with atrazine. The efficiency of the immunofluorescence procedure was compared using several antisera obtained by immunizing with different hapten conjugates and purified by various purification methods. The best results were observed with the atrazine analogue ametryn sulfoxide, which was coupled to bovine serum albumin for immunization and to Sepharose for immunoaffinity chromatography. The procedure described in this paper may serve as a general tool for detecting bound pesticide residues in plant material. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

A real-time immuno-PCR assay for routine ultrasensitive quantification of proteins

A fast and robust assay, based on the combination of the highly sensitive immuno-PCR (IPCR), employing standardized self-assembled DNA-protein conjugates as reagents, and the well-established, reliable, and fast real-time PCR detection by means of the TaqMan principle is introduced in this work. The use of anti-species immunoglobulin reagents allows one for easy adaptation of this assay to basically any existing ELISA application. The use of an internal competitor in the real-time IPCR (rtIPCR) further increases the sensitivity and significance of this assay; 0.1-0.01 amol (500-50 fg/mL) IgG from several species (mouse, rabbit, goat, and human) were detectable using direct, indirect, and sandwich model rtIPCR assays, thereby increasing the detection limit of the analogous ELISA tests about 100- to 1000-fold. The robustness of this method was demonstrated in two typical applications by detecting 40 pg/mL of the novel anti-cancer drug rViscumin in human plasma samples as well as 100 pg/mL of a research antibody in cell culture media. In both cases, a comparable ELISA was 1000-fold less sensitive.     

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Linear-amplification mediated PCR (LAM-PCR) has been developed to study hematopoiesis in gene corrected cells of patients treated by gene therapy with integrating vector systems. Due to the stable integration of retroviral vectors, integration sites can be used to study the clonal fate of individual cells and their progeny. LAM- PCR for the first time provided evidence that leukemia in gene therapy treated patients originated from provirus induced overexpression of a neighboring proto-oncogene. The high sensitivity and specificity of LAM-PCR compared to existing methods like inverse PCR and ligation mediated (LM)-PCR is achieved by an initial preamplification step (linear PCR of 100 cycles) using biotinylated vector specific primers which allow subsequent reaction steps to be carried out on solid phase (magnetic beads). LAM-PCR is currently the most sensitive method available to identify unknown DNA which is located in the proximity of known DNA. Recently, a variant of LAM-PCR has been developed that circumvents restriction digest thus abrogating retrieval bias of integration sites and enables a comprehensive analysis of provirus locations in host genomes. The following protocol explains step-by-step the amplification of both 3’- and 5’- sequences adjacent to the integrated lentiviral vector.     

Early emergence increases survival of tree seedlings in Central European temperate forests despite severe late frost

Global warming is expected to result in earlier emergence of tree seedlings that may experience higher damages and mortality due to late frost in spring. We monitored emergence, characteristics, and survival of seedlings across ten tree species in temperate mixed deciduous forests of Central Europe over one and a half year. We tested whether the timing of emergence represents a trade‐off for seedling survival between minimizing frost risk and maximizing the length of the growing period. Almost two‐thirds of the seedlings died during the first growing period. The timing of emergence was decisive for seedling survival. Although seedlings that emerged early faced a severe late frost event, they benefited from a longer growing period resulting in increased overall survival. Larger seedling height and higher number of leaves positively influenced survival. Seedlings growing on moss had higher survival compared to mineral soil, litter, or herbaceous vegetation. Synthesis. Our findings demonstrate the importance of emergence time for survival of tree seedlings, with early‐emerging seedlings more likely surviving the first growing period.     

The size of the photosynthetic unit in purple bacteria

Pigment analysis was performed by means of normal phase HPLC on a number of bacteriochlorophyll a and b containing species of purple bacteria that contain a core antenna only. At least 99% of the bacteriochlorophyll in Rhodobacter sphaeroides R26, Rhodopseudomonas viridis and Thiocapsa pfennigii was esterified with phytol (BChl a _p and BChl b _p, respectively). Rhodospirillum rubrum contained only BChl a esterified with geranyl-geraniol (BChl a _GG). Rhodospirillum sodomense and Rhodopseudomonas marina contained, in addition to BChl a _p, small amounts of BChl a _GG, and presumably also of BChl a esterified with dihydro and tetrahydro geranyl-geraniol (2,10,14-phytatrienol and probably 2,14-phytadienol). In all species bacteriopheophytin (BPhe) esterified with phytol was present. The BChl/BPhe ratio indicated that in these species a constant number of 25 ± 3 antenna BChls is present per reaction centre. This number supports a model in which the core antenna consists of 12 - heterodimers surrounding the reaction centre. Determination of the in vivo extinction coefficient of BChl in the core-reaction centre complex yielded a value of ca. 140 mM^–1 cm^–1 for BChl a containing species and of 130 mM^–1 cm^–1 for Rhodopseudomonas viridis.Abbreviations BChl bacteriochlorophyll - BPhe bacteriopheophytin - GG geranyl-geraniol - LHI and LHII core and peripheral antenna complexes - P phytol - RC reaction centre Dedicated to the memory of Professor D.I. Arnon.