Canine Haematopoietic Chimerism Analyses by Semiquantitative Fluorescence Detection of Variable Number of Tandem Repeat Polymorphism

Canine models are successfully applied to the study of haematopoietic stem cell transplantation (HSCT). Monitoring of haematopoietic donor/recipient chimerism is of major significance in detecting and quantifying engraftment or graft rejection of the donor-derived haematopoietic cells after transplantation. Radioactive analyses of polymorphic microsatellite markers are commonly used for chimerism analyses. We describe an improved, non-isotopic method that is based on the analysis of microsatellite markers in donor and recipient cells using capillary electrophoresis and fluorescence detection. Artificial mixtures of donor and recipient DNA that were generated from peripheral blood mononuclear cells from dog leukocyte antigen-identical siblings were used to analyse the sensitivity of the assay. DNA from dogs that had received HSCT were also analysed in order to demonstrate the feasibility of the method in vivo. For chimerism analyses, six different microsatellite loci were systematically amplified using fluorescent PCR primer. The fluorescent polymerase chain reaction products were separated by capillary electrophoresis using POP4 on a 310 ABI Prism Genetic Analyzer. After electrophoresis, fluorescence signals were automatically sized and quantified using GeneScan software. The method described provides an accurate assessment of haematopoietic chimerism in the canine model with significantly reduced hands-on time compared to conventional gel electrophoresis.     

A Comparative Study of Staphylococcus aureus Strains Isolated from Bovine Subclinical Mastitis During 1952–1956 and 1992

Fiftytwo strains of S. aureus isolated from cases of bovine subclinical mastitis in 52 different dairy herds in Denmark, in the periods 1952 to 1956 and 1992, were compared with regard to their phage- and EcoRI ribo-types. Furthermore, susceptibility to penicillin and production of fibrinolysin were used as additional phenotypic markers. Fortynine strains (94%) could be separated into 12 phage types. Ribotyping assigned the 52 strains to 21 different types. Both methods showed that 57% of the 1950’s strains and between 38–45% of the 1992 strains belonged to 3 dominating types. The remaining strains were placed by ribotyping in 8 types occurring among the 1952–1956 strains and 10 types occurring among the 1992 strains. In 87% of the strains the results of the 2 typing methods were in accordance. However, 7 strains gave different results by the 2 methods including 2 strains with major differences. Penicillin resistance only occurred in a single genotype from the 1950’s compared to 6 different genotypes among the 1992 strains.     

Second-harmonic generation from magnetic metamaterials

We observe second-harmonic generation from metamaterials composed of split-ring resonators excited at 1.5-micrometer wavelength. Much larger signals are detected when magnetic-dipole resonances are excited, as compared with purely electric-dipole resonances. The experiments are consistent with calculations based on the magnetic component of the Lorentz force exerted on metal electrons-an intrinsic second-harmonic generation mechanism that plays no role in natural materials. This unusual mechanism becomes relevant in our work as a result of the enhancement and the orientation of the local magnetic fields associated with the magnetic-dipole resonances of the split-ring resonators.     

Characterization of different wood samples using a new combined method of evolved gas analysis and pyrolysis–gas chromatography/mass spectrometry

Evolved gas analysis (EGA) in combination with analytical pyrolysis is presented as a new technique for rapid investigations into wooden materials. In a first analytical step, the thermal behaviour of three wood samples was determined by EGA. Based on these results, analytical pyrolysis–gas chromatography/mass spectrometry was carried out to investigate the influence of wood-specific thermal behaviour and the chemical composition on the results. Furthermore, the influence of sample mass and particle size was examined.     

甘蓝型油菜(Brassica napus)无花瓣突变体“ Ap-Tengbe ”花瓣性状的遗传规律

本项研究旨在探明甘蓝型油菜突变体Ａｐ－Ｔｅｎｇｂｅ无花瓣性状的遗传规律,‘Ａｐ－Ｔｅｎｇｂｅ’和德国栽培品种‘Ｆａｌｃｏｎ’之间通过正反交产生两个方向的Ｆ１,ＢＣ１－１（Ｆ１和‘Ａｐ－Ｔｅｎｇｅｂｅ’之间）,ＢＣ１－２（Ｆ１和‘Ｆａｌｃｏｎ’之间）以及Ｆ２各遗传群体,根据田间各遗传群体ＰＤｇｒ（花瓣度）的观察和统计,作者提出‘Ａｐ－Ｔｅｎｇｅ’突变体无花瓣性状的遗传由细胞质和两对核隐性基因共同控制     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

Inheritance of a pectate lyase mediated soft rot resistance in crosses between transgenic potato lines of cv. désirée andS. tuberosum cultivars

Summary Expression of the pectate lyase (PL) isoenzyme 3 in transgenic potato lines of cv. Désirée mediates an enhanced resistance of tuber tissue toErwinia carotovora (Ec) soft rot due to a pre-activation of plant defence mechanisms. Therefore, theSolanum tuberosum cvs Agave and Adretta with a moderate level of soft rot resistance were crossed with such PL-expressing potato lines. The resulting progenies were assessed with respect to plant/tuber characteristics over a period of four years and then tested for PL3-expression as well as for soft rot resistance. 71% of the selected progeny lines exhibited a stable production of the PL3 enzyme. Statistical analysis revealed differences between the transgenic and the non-transgenic progeny concerning the soft rot resistance of tuber tissue. Compared with the PL-inactive progeny, extension of Ec-rotting on the wound surface of PL-transgenic potatoes was diminished on average by 51.8%. Similarly, the degree of cell lysis caused by bacterial maceration was significantly (P<0.05) reduced in tuber tissue of PL-expressing progeny lines. The latter also revealed an enhanced PPO and PAL activity in their tuber tissue indicating an active plant defence. It is concluded therefore that the PL-mediated soft rot resistance introduced into potatoes by means of molecular techniques is heritable.