Steric aspects of agonism and antagonism at beta-adrenoceptors: synthesis of and pharmacological experiments with the enantiomers of formoterol and their diastereomers.

The enantiomers of formoterol (R;R and S;S) and their diastereomers (R;S and S;R) were synthesized and purified using a new procedure which required the preparation of the (R;R)- and (S;S)-forms of N-(1-phenylethyl)-N-(1-(p-methoxyphenyl)-2-propyl)-amine as important intermediates. The enantiomeric purity obtained was greater than 99.3%, usually greater than 99.7%. The four stereoisomers were examined with respect to their ability to interact in vitro with beta-adrenoceptors in tissues isolated from guinea pig. The effects measured were (1) relaxation of the tracheal smooth muscle (mostly beta 2), (2) depression of subtetanic contractions of the soleus muscle (beta 2), and (3) increase in the force of the papillary muscle of the left ventricle of the heart (beta 1). All enantiomers caused a concentration-dependent and complete relaxation of the tracheal smooth muscle which was inhibited by propranolol. The order of potency was (R;R) much greater than (R;S) = (S;R) greater than (S;S). There was a 1,000-fold difference in potency between the most and the least potent isomer. The presence of the (S;S)-isomer did not affect the activity of the (R;R)-isomer on the tracheal smooth muscle. Also on the skeletal and cardiac muscles (R;R)-formoterol was more potent than its (R;S)-isomer. The selectivity for beta 2-adrenoceptors appeared to be slightly higher for the (R;R)-isomer than for the (R;S)-isomer. The potency of the (S;R)- and (S;S)-isomers on the papillary muscle was too low to be determined accurately.(ABSTRACT TRUNCATED AT 250 WORDS)     

Steric aspects of formoterol and terbutaline: Is there an adverse effect of the distomer on airway smooth muscle function?

Experiments were made on isolated tissues from guinea-pig to test the hypothesis that the distomers of rac-β₂-adrenoceptor agonists induce airway hyperreactivity. Tracheal strip preparations were contracted with carbachol. Both rac- and (R;R)-formoterol (2 and 1 μmol/1, respectively) produced an immediate relaxation, followed by a slow recovery of tone. (S;S)-Formoterol (2 μmol/1) had no effect on smooth muscle tone. Similar results were obtained with the enantiomers of terbutaline. In other strip preparations of the trachea or the main bronchi, cholinergic or nonadrenergic/noncholinergic (NANC) excitatory responses were evoked by electrical field-stimulation. The eutomers, (R;R)-formoterol and (R)-terbutaline, inhibited concentration-dependently both cholinergic and NANC-induced contractions. The distomers, (S;S)-formoterol and (S)-terbutaline, showed qualitatively the same effects but were about 1,000 times less potent than the corresponding eutomer. In a third series of experiments, either enantiomer of formoterol was administered to an electrically stimulated vagus nerve-trachea tube preparation. The nerve-induced contractions were inhibited by both enantiomers, but (S;S)-formoterol was about 1,000 times less potent than (R;R)-formoterol. For both enantiomers of formoterol, about tenfold higher concentration was required to obtain the same degree of inhibition when given intratracheally as compared with administration in the external medium. There was no indication in any of the experimental approaches that (S;S)-formoterol or (S)-terbutaline might enhance the response to cholinergic or NANC-related stimuli. Chirality 8:567–573, 1996. © 1997 Wiley-Liss, Inc.     

Efficient dsRNA-mediated transgenic resistance to Beet necrotic yellow vein virus in sugar beets is not affected by other soilborne and aphid-transmitted viruses

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is one of the most devastating sugar beet diseases. Sugar beet plants engineered to express a 0.4 kb inverted repeat construct based on the BNYVV replicase gene accumulated the transgene mRNA to similar levels in leaves and roots, whereas accumulation of the transgene-homologous siRNA was more pronounced in roots. The roots expressed high levels of resistance to BNYVV transmitted by the vector, Polymyxa betae. Resistance to BNYVV was not decreased following co-infection of the plants with Beet soil borne virus and Beet virus Q that share the same vector with BNYVV. Similarly, co-infection with the aphid-transmitted Beet mild yellowing virus, Beet yellows virus (BYV), or with all of the aforementioned viruses did not affect the resistance to BNYVV, while they accumulated in roots. These viruses are common in most of the sugar beet growing areas in Europe and world wide. However, there was a competitive interaction between BYV and BMYV in sugar beet leaves, as infection with BYV decreased the titres of BMYV. Other interactions between the viruses studied were not observed. The results suggest that the engineered resistance to BNYVV expressed in the sugar beets of this study is efficient in roots and not readily compromised following infection of the plants with heterologous viruses.     

dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris)

Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties.     

QTL mapping of BNYVV resistance from the WB41 source in sugar beet.

The most important rhizomania-resistance gene in sugar beet is the Rz1 gene from the Holly Sugar Company in California, the source widely used to breed partially resistant varieties. Other important gene sources are WB41 and WB42, which both originate from Beta vulgaris subsp. maritima collected in Denmark, and which have been reported to be similar. The major resistance gene in WB42 is known as Rz2. We studied the resistance in WB41 and used markers to map the major resistance gene in this source, which we call Rz3. It was identified on chromosome III. This is the chromosome that Rz1 and Rz2 have been mapped to. Data from greenhouse tests and ELISA showed that Rz3 had incomplete penetrance, with heterozygotes varying widely in resistance levels. The involvement of additional minor genes in the strong resistance of the original WB41 source cannot be excluded.     

Evaluation of a Direct Agglutination Test for Detection of Antibodies Against Toxoplasma gondii in Cat,Pig and Sheep Sera

The protozoan parasite Toxoplasma gondii is the causative agent of the zoonosis toxoplasmosis. In sheep and goats, it is one of the most prevalent causes of infectious abortion. Also in pregnant women, a primary infection can result in miscarriage. Humans acquire the infection either by ingestion of oocysts excreted by cats, the definitive host of the parasite, or by eating raw or undercooked meat from latently infected animals (Dubey & Beattie 1988). In Sweden, toxoplasmosis is a notifiable disease, and cases of clinical disease in humans as well as animals must be reported. In both veterinary and human medicine serological assays based on detecting the humoral antibody response of the host against the parasite are used as diagnostic tools. So far, solid phase assays, such as the indirect fluorescent antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA), have been widely used to diagnose T. gondii infection in many species including cats, pigs and sheep (Dubey & Beattie 1988). However, both IFAT and ELISA require appropriate anti-species specific immunoglobulins (Ig) that must be carefully evaluated for each species prior to use. This makes these assays complicated and time consuming. Consequently, alternative, simpler methods that do not require specific antisera would be of great value. The direct agglutination test (DA), which is based on the principle that formalin-treated organisms agglutinate in the presence of specific IgG antibodies, is such an assay (Fulton & Turk 1959). The DA-test is widely used in human medicine as a screening test for T gondii infection but it has not yet been thoroughly evaluated for use in veterinary medicine (Uggla & Buxton 1990).