RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.     

Automated assay of phosphohydrolases by measuring the released phosphate without deproteinization.

We describe an automated determination of inorganic phosphate in the presence of proteins and its application for the assay of NaK-ATPase (EC 3.6.1.3) and other insoluble phosphohydrolases. After incubation the enzyme reaction is stopped at neutral pH with 3.3% (w/v) sodium dodecyl sulfate plus 30 mm EDTA (final concentration). The released phosphate is measured on the Technicon Autoanalyzer as phosphomolybdate reduced with ferrous ammonium sulfate and thiourea to molybdenum blue. EDTA enhances color development and solubilization of the proteins. The reagents are stable at room temperature and are formulated from cheap, common chemicals.     

Determination of Wolbachia genome size by pulsed-field gel electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.     

Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia

Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.     

Real-time PCR for quantification of the bacterial endosymbionts (Wolbachia) of filarial nematodes

Filarial nematodes harbour intracellular symbiotic bacteria belonging to the genus Wolbachia. Wolbachia is thought to play an important role in the biology of the nematode. Moreover, Wolbachia appears to be involved in the immunopathogenesis of filariasis and in the onset of the side-effects of antifilarial therapy. Investigations in these research areas require reliable methods to quantify Wolbachia both in nematodes and in vertebrate tissues. To this purpose, we designed a quantitative real-time PCR targeted on the ftsZ gene of the Wolbachia of Brugia pahangi, a model filarial species maintained in gerbils. The method was applied to quantify Wolbachia in Brugia pahangi, from animals with or without tetracycline treatment. Our results show that tetracycline treatment leads to dramatic reduction or clearance of Wolbachia from the nematode. Results obtained from different replicates were reproducible and the method appeared very sensitive compared to other PCR protocols for Wolbachia detection. Real-time PCR is thus an appropriate method for investigations on the biological role of Wolbachia and on the implication of these bacteria in the pathogenesis of filariasis. With slight modifications of the primers and probe, the protocol we have developed could be applied in studies of the human pathogen Brugia malayi and on the model filarial species Litomosoides sigmodontis.     

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.