Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable.     

In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy

Whereas the morphogenesis of developing organisms is relatively well understood at the molecular level, the contribution of the mechanical properties of the cells to shape changes remains largely unknown, mainly because of the lack of quantified biophysical parameters at cellular or subcellular resolution. Here we designed an atomic force microscopy approach to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAMs). SAMs are highly organized structures that contain the plant stem cells, and generate all of the aerial organs of the plant. Building on modeling and experimental data, we designed a protocol that is able to measure very local properties, i.e. within 40-100 nm deep into the wall of living meristematic cells. We identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and even subcellular levels. Strikingly, we found that the outer cell wall was much stiffer at the tip of the meristem (5 ± 2 MPa on average), covering the stem cell pool, than on the flanks of the meristem (1.5 ± 0.7 MPa on average). Altogether, these results demonstrate the existence of a multiscale spatialization of the mechanical properties of the meristem surface, in addition to the previously established molecular and cytological zonation of the SAM, correlating with regional growth rate distribution.     

Do synovial biopsies help to support evidence for involvement of innate immunity in the immunopathology of Beh?et's disease?

Behçet's disease is a complex vasculitis of unknown etiology. Abundant neutrophils suggest the involvement of innate immunity. Cytokines are skewed to the T-helper-1 pattern. Few sterile organs are easily accessible for analysis in Behçet's disease. Cañete and coworkers identify inflamed joints as a feasible model and suggest the involvement of innate immunity in Behçet's disease.     

Amyloid induced suicidal erythrocyte death.

Amyloid peptides are known to induce apoptosis in a wide variety of cells. Erythrocytes may similarly undergo suicidal death or eryptosis, which is characterized by scrambling of the cell membrane with subsequent exposure of phosphatidylserine (PS) at the cell surface. Eryptosis is triggered by increase of cytosolic Ca(2+) activity and by activation of acid sphingomyelinase with subsequent formation of ceramide. Triggers of eryptosis include energy depletion and isosmotic cell shrinkage (replacement of extracellular Cl(-) by impermeable gluconate for 24 h). The present study explored whether amyloid peptide Abeta (1-42) could trigger eryptosis and to possibly identify underlying mechanisms. Erythrocytes from healthy volunteers were exposed to amyloid and PS-exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca(2+) activity (Fluo3 fluorescence) and ceramide formation (anti-ceramide antibody) were determined by FACS analysis. Exposure of erythrocytes to the amyloid peptide Abeta (1-42) (> or = 0.5 microM) for 24 h significantly triggered annexin V binding, an effect mimicked to a lesser extent by the amyloid peptide Abeta (1-40) (1 microM). Abeta (1-42) (> or = 1.0 microM) further significantly decreased forward scatter of erythrocytes. The effect of Abeta (1-42) (> or = 0.5 microM) on erythrocyte annexin V binding was paralleled by formation of ceramide but not by significant increase of cytosolic Ca(2+) activity. The presence of Abeta (1-42) further significantly enhanced the eryptosis following Cl(-) depletion but not of glucose depletion for 24 hours. The present observations disclose a novel action of Abeta (1-42), which may well contribute to the pathophysiological effects of amyloid peptides, such as vascular complications in Alzheimer's disease.     

Immunogold labeling of cryosections from high-pressure frozen cells

Immunogold labeling of cryosections according to Tokuyasu (Tokuyasu KT. A technique for ultracyotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–565), is an important and widely used method for immunoelectron microscopy. These sections are cut from material that is chemically fixed at room temperature (room temparature fixation, RTF). Lately in many morphological studies fast freezing followed by cryosubstitution fixation (CSF) is used instead of RTF. We have explored some new methods for applying immunogold labeling on cryosections from high‐pressure frozen cells (HepG2 cells, primary chondrocytes) and tissues (cartilage and exocrine pancreas). As immunolabeling has to be carried out on thawed and stable sections, we explored two ways to achieve this: (1) The section fixation method, as briefly reported before (Liou W et al. Histochem Cell Biol 1996;106:41–58 and Möbius W et al. J Histochem Cytochem 2002;50:43–55.) in which cryosections from freshly frozen cells were stabilized in mixtures of sucrose and methyl cellulose and varying concentrations of glutaraldehyde, formaldehyde and uranyl acetate (UA). Only occasionally does this method reveal section areas with excellent cell preservation and negatively stained membranes like Tokuyasu sections of RTF material. (Liou et al.) (2) The rehydration method, a novel approach, in which CSF with glutaraldehyde and/or osmium tetroxide (OsO₄) was followed by rehydration and cryosectioning as in the Tokuyasu method. Especially, the addition of UA and low concentrations of water to the CSF medium favored superb membrane contrast. Immunogold labeling was as efficient as with the Tokuyasu method.     

Subthalamic nucleus deep brain stimulation in elderly patients – analysis of outcome and complications

Background  

There is an ongoing discussion about age limits for deep brain stimulation (DBS). Current indications for DBS are tremor-dominant disorders, Parkinson's disease, and dystonia. Electrode implantation for DBS with analgesia and sedation makes surgery more comfortable, especially for elderly patients. However, the value of DBS in terms of benefit-risk ratio in this patient population is still uncertain.     

Myelin basic protein purified on an ion-exchange continuous polymer bed in the presence of ethylene glycol and salt possesses activity againstp-nitrophenyl acetate

In this paper we describe a fast and mild method based on the use of a unique cation exchanger and buffers containing ethylene glycol and salt for the purification of the myelin basic protein (MBP; MW 18.5 kDa). MBP thus purified hydrolyses catalytically p-nitrophenyl acetate. This esterase activity facilitates not only the purification of MBP but also indicates that probably it is in its native state, i.e. there is a good chance that the purified molecules are structurally and chemically identical. This is a prerequisite to obtain crystals appropriate for x-ray diffraction and other studies.Abbreviations used MBP myelin basic protein - MW molecular weight - kDa kilo Dalton - octyl-POE n-octylpolydisperse oligooxyethylene - CHAPS 3-3-cholamidopropyl dimethylammonio-1-propane-sulfonate - CTAB cetyltrimethylammonium bromide - SDS sodium dodecyl sulfate - SDS-PAGE polyacrylamide gel electrophoresis in the presence of SDS - G 3707 heptaoxyethylene lauryl ether - TWEEN-20 polyoxyethylenesorbitan-monolaurat - EDTA ethylenediaminetetraacetic acid - HEPES N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid)     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Reproductive strategies of the orang-utan: New data and a reconsideration of existing sociosexual models

Recent field data indicate that MacKinnon’s model of the orang-utan’s sexual and agonistic activity needs to be revised. In this model, male reproductive activity is concentrated in an extended phase of subadulthood and in early adulthood. According to this model, the role of older adult males is primarily that of range guardian, and in that role they would ensure that the offspring they had generated earlier would have safe access to food resources. This study presents cases suggesting that subadult males, even though sexually active, may have low reproductive success. In previous studies adult males were shown to display less sexual initiative than subadult males. In this study an adult male was at times involved infrequent mating activity in response to proceptive activity of females in the course of consortship. This adult male proved to be a successful breeder, thus refuting the hypothesis of adult male sterility. The female is most likely to conceive through cooperative mating in lengthy consortships with the dominant resident adult male. We hypothesize that the extended subadult phase represents a submissive strategy, allowing subadult males to remain in the home range of adult males but with minimal reproductive success.