Hepatitis E virus in wild rabbits and European brown hares in Germany.

Recently, a change of hepatitis E from being a typical travel-associated disease to an autochthonous zoonosis in Germany was observed. An increasing number of autochthonous infections with the hepatitis E Virus (HEV) have been recognized in developed countries. Venison from wild boar is already known to be a potential source of infection, if not prepared properly by the consumer. In Germany, certain wild animals are known to be a reservoir for HEV. However, current information is missing about European brown hares (Lepus europaeus) and wild rabbits (Oryctolagus cuniculus). Thus, a total of 833 hunting-harvested animals (European brown hares n = 669; wild rabbits n = 164) were tested for the occurrence of HEV RNA and HEV antibodies. For this, liver and blood specimens were taken after hunts in six German federal states. HEV antibodies were found by ELISA in 2.2% (624/14) of European brown hares, but no HEV RNA was detectable by nested real-time RT-PCR. In contrast, a seroprevalence of 37.3% (126/47) was observed for wild rabbits, and 17.1% (164/28) of the samples were HEV RNA positive. Genomic analysis revealed that these partial sequences clustered within the rabbit clade of HEV-3 genotype. In addition, one rabbit sequence segregated into subtype 3g of HEV-3. Highest seroprevalences for hares and rabbits were detected in the federal states of Bavaria and of Schleswig-Holstein, respectively. Comparing urban, rural and insular areas, the highest seroprevalence was shown for wild rabbits in rural areas and for European brown hares on the northern island Fehmarn. This study provides evidence that European brown hares and wild rabbits from Germany can be infected with HEV. The different prevalences indicate that wild rabbits are a potential reservoir for HEV in Germany, whereas European brown hares seem to be only of minor importance for the epidemiology of HEV.

Towards the biodegradation pathway of fosfomycin.

Three functionalised propylphosphonic acids were synthesised to study C-P bond cleavage in R. huakuii PMY1. (R)-1-Hydroxy-2-oxopropylphosphonic acid [(R)-5] was prepared by chiral resolution of (±)-dimethyl 1-hydroxy-2-methylallyllphosphonate [(±)-12], followed by ozonolysis and deprotection. The N-(l-alanyl)-substituted (1R,2R)-2-amino-1-hydroxypropylphosphonic acid 10, a potential precursor for 2-oxopropylphosphonic acid (5) in cells, was obtained by coupling the aminophosphonic acid with benzotriazole-activated Z-l-alanine and hydrogenolytic deprotection. (1R*,2R*)-1,2-Dihydroxy-3,3,3-trifluoropropylphosphonic acid, a potential inhibitor of C-P bond cleavage after conversion into its 2-oxo derivative in the cell, was accessed from trifluoroacetaldehyde hydrate via hydroxypropanenitrile 21, which was silylated and reduced to the aldehyde (±)-23. Diastereoselective addition of diethyl trimethylsilyl phosphite furnished diastereomeric α-siloxyphosphonates. The less polar one was converted to the desired racemic phosphonic acid (±)-(1R*,2R*)-9 as its ammonium salt.

Aspects of 6-[18F]fluoro-L-DOPA preparation: precursor synthesis, preparative HPLC purification and determination of radiochemical purity.

A modified method for the synthesis of the intermediate product N-Boc-3,4-di(Boc-O)-6-iodo-L-phenylalanine ethyl ester of the [18F]FDOPA precursor preparation was developed. With the application of bis-(trifluoroacetoxy)-iodobenzene for the iodination step with elemental iodine the yield of the intermediate can be increased from 12% to 50-60%. By replacing silica-gel-based RP HPLC column by a polymer-based column for semi-preparative purification of [18F]FDOPA from the reaction mixture the radiochemical purity of the final product can be increased up to >99%. For the determination of the radiochemical impurity [18F]fluoride a HPLC method using a column with polymer-based RP material was introduced.

Simultaneous separation of the stereoisomers of 1-amino-2-hydroxy and 2-amino-1-hydroxypropane phosphonic acids by stereoselective capillary electrophoresis employing a quinine carbamate type chiral selector.

A stereoselective nonaqueous capillary electrophoresis (CE) method utilizing O-(tert-butylcarbamoyl) quinine as chiral ion-pair agent and additive to the non aqueous background electrolyte was evaluated for the simultaneous separation of the enantiomers and diastereomers of 1 -amino-2-hydroxypropane phosphonic acid besides the corresponding beta-aminophosphonic acid analogs, the stereoisomers of 2-amino-1-hydroxypropane phosphonic acid, in a single run. The separations have been carried out using the partial filling technique to avoid strong background signal from the quinine selector. It conveniently allowed the baseline separation of all eight components of interest (alpha- as well as beta-aminophosphonic acids) as N-2,4-dinitrophenyl derivatives in a single run. Moreover, the absolute configurations of all eight peaks were identified. Compared to the quinine carbamate selector, the corresponding 'pseudo-enantiomeric' O-(tert-butylcarbamoyl) quinidine selector exhibited reserved elution order and nearly identical resolutions. The proposed CE method turned out to be advantageous over stereoselective high-performance liquid chromatography (HPLC) with a quinine carbamate type stationary phase, which showed high enantioselectivity, but failed to simultaneously separate all eight components.

Transformation of arylmethylamines into alpha-aminophosphonic acids via metalated phosphoramidates: rearrangement of partly configurationally stable N-phosphorylated alpha-aminocarbanions

N-Benzyl phosphoramidate was protected at nitrogen with a TMS, p-toluenesulfonyl, Boc, lithium carboxylate, or diethoxyphosphinyl group and metalated with s-BuLi or LDA at -78 degrees C at the benzylic carbon. For the latter three protecting groups, the intermediate alpha-amino(phenylmethyl)lithiums isomerized to N-protected alpha-aminophosphonates (phosphoramidate-aminophosphonate rearrangement). (R)-N-[1-(2)H(1)]Phenylmethyl phosphoramidate in combination with Boc or (EtO)(2)P(O) was used to demonstrate that metalation occurs with a high primary kinetic isotope effect (k(H)/k(D) 13-50) and migration of the diethoxyphosphinyl group with retention of configuration at carbon. Furthermore, the short-lived carbanion lithium pairs are partly configurationally stable as the aminophosphonates formed with the two protecting groups have enantiomeric excesses of 79 and 24%, respectively. When homochiral lithium amides derived from (R)-N-isopropyl-1-phenylethylamine and (R,R)-N,N-di(1-phenylethyl)amine were used to induce a phosphoramidate-aminophosphonate rearrangement, chiral nonracemic alpha-aminophosphonates were formed (ee 26-35%). Three racemic aminophosphonates were deprotected with hot 6 M HCl and purified by ion-exchange chromatography on Dowex 50W,H(+) to exemplify the transformation of phenyl-, p-tolyl-, and (1'-naphthyl)methylamine into aminophosphonic acids via lithiated phosphoramidates.

In vitro degradation of atracurium and cisatracurium at pH 7.4 and 37 degrees C depends on the composition of the incubating solutions.

The pharmacokinetic models proposed for atracurium or cisatracurium are based on the assumption that spontaneous degradation via Hofmann elimination proceeds in vivo at the same rate as measured in vitro at pH 7.4 and 37 degrees C. As different degradation rates have been reported for all 10 stereoisomers of atracurium measured together, for each of its three isomeric groups, and for the single isomer cisatracurium, we studied if the rate is dependent on factors other than pH and temperature. In vitro degradation of atracurium and cisatracurium was studied at 37 degrees C and pH 7.4 in nine incubating solutions containing one of three buffer systems (phosphate, HEPES or Tris) and additives (sodium chloride, potassium sulphate or glucose). Concentrations of atracurium, cisatracurium and laudanosine were measured after incubation for up to 240 min using an HPLC method. Degradation of atracurium proceeded monoexponentially. The rate was slower in the presence of sodium chloride, potassium sulphate, and in a lower concentration of the phosphate buffer. Glucose enhanced the degradation. At the same total buffer concentration (50 mmol litre-1), degradation was fastest in the phosphate, intermediate in the HEPES and slowest in the Tris buffer. Degradation rates of cisatracurium in sodium phosphate 50 mmol litre-1 and Sörensen (Na-K phosphate) buffer 66.7 mmol litre-1 were similar to those of atracurium. We conclude that, at constant pH and temperature, the degradation rate of atracurium was dependent on the total concentration of the base in the incubating solution.

Biodegradation of phosphonomycin by Rhizobium huakuii PMY1.

The biodegradation by Rhizobium huakuii PMY1 of up to 10 mM phosphonomycin as a carbon, energy, and phosphorus source with accompanying P(i) release is described. This biodegradation represents a further mechanism of resistance to this antibiotic and a novel, phosphate-deregulated route for organophosphonate metabolism by Rhizobium spp.

Stereochemistry of the reaction catalysed by 2-aminoethylphosphonate aminotransferase. A 1H-NMR study.

(R)- and (S)-2-amino[2-D1]ethylphosphonic acids ([2-D1]AEP) were synthesised to investigate the stereochemistry of the reaction catalysed by 2-aminoethlphosphonate aminotransferase from Pseudomonas aeruginosa. This enzyme catalyses the transfer of the amino group of AEP to pyruvate to produce 2-phosphonoacetaldehyde and alanine. The enzymic reaction proceeding through the abstraction of a proton from the Schiff-base complex formed between the enzyme-bound pyridoxal 5'-phosphate, and the substrate, was carried out in an aqueous buffer at pH 8.5; it was followed by high-field 1H-NMR measurements (500 MHz, H2O) on an AMX 500 Bruker spectrometer. The spectra, recorded with chiral (R)- or (S)-[2-D1]AEP, both showed the methylenic signal (3.0 ppm), whereas (S)-[2-D1]AEP gave the additional aldehydic signal (CHO, 9.6 ppm). These data clearly show that AEP-aminotransferase catalyses the abstraction of the pro-S hydrogen atom at the prochiral C2 carbon of AEP. Furthermore, careful timing of NMR measurements over a 2-hour period allows us to show the occurrence of an isotopic effect.

Chemical composition and antimicrobial activity of essential oils of Jasonia candicans and J. montana.

The essential oils of the aerial parts of Jasonia candicans and J. montana were analyzed by gas chromatography-mass spectrometry (GC/MS) technique. Of twenty-one components identified in the volatile oil of J. candicans, intermediol was the main constituent. Fifty-eight components were characterized in the essential oil of J. montana. Camphor, borneol, bornyl acetate, chrysanthemol, intermediol, and 1,8-cineole were the major constituents in this oil. The two oils showed antibacterial activity against Bacillus subtilis. They also showed a marked antifungal activity against Trichophyton mentagrophytes, Cryptococcus neoformans, and Candida albicans.