Comparative analysis of ammonia monooxygenase (amoA) genes in the water column and sediment-water interface of two lakes and the Baltic Sea

The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Schöhsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Schöhsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Schöhsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environment

ABBREVIATIONS: CNS, centrainervoussystem;R-PIA,N6-[R-(--)-1-methyl-2-phenethyljadenosine; NECA, 5'-N-ethylcarboxamidoadenosine; PV, purifiedmyentencvancosities; LDH,Lactatedehydrogenase; Affinity of VariousPurineNucleosides forAdenosine Receptors on Purif

ABSTRACT Isolated myentenc varicosities (autonomic synaptosomes) pre pared from the guinea-pig ileum were used as the substrate in competition experiments designed to study the properties of the adenosine receptor present on peripheral nerve endings. Com petition experiments using [3HJ-N6-[R-(â€")-1 -methyl-2-phenethylj adenosine and [3H]-5'-N-ethylcarboxamidoadenosine as the Ia beled ligands permifted the affinities of several unlabeled aden osine analogs to be examined. In addition, the ability of theo phylline, an antagonist at adenosine receptors, to compete for binding was determined. The relative affinities of the nucleosides were compared to their efficacies as inhibitors of acetylcholine release in the stimulated guinea-pig ileum preparation and an excellent correlation was obtained. The identity of the substrate used in the binding studies with the target in the bioassay system suggests that the isolated myentenc vancosity contains the adenosine receptor responsible for the observed biological activ ity. Similaraffinities for theophylline as a competitor of the binding of both labeled ligands paralleled the establishment of similar PA2 values for the antagonist in the bioassay. These findings, together with the similarity of the biological efficacy of N6-[R-(â€")-1-methyl-2-phenethyljadenosine and 5'-N-ethylcarboxamidoa denosine, suggest that the adenosine receptor present on myen tenc nerve endings is unitary but do not permit its designation as an A1or A2subtype. On the basis of the interaction of adenine nucleosides with adenylate cyclase, two adenosine receptor subtypes have been proposed. The designation of A1 and A2 receptor subtypes proposed by van Calker et at. (1979) resulted from observation of a concentration-dependent selectivity of the activity of aden osine in inhibiting (A1) or stimulating (A2) the enzyme. The use of specific analogs has reinforced this classification (for reviews see Berne et at., 1983) and N6-substituted compounds such as R-PIA and C5'-substituted compounds such as NECA have become established as prototypical A1 and A2 receptor agonists respectively. Although the involvement of adenylate cyclase in the inhib itory response to adenosine at the CNS is not clear, Kuroda et at. (1976) have described the stimulatory effect ofthe nucleoside in the cyclase of guinea-pig cortical slices in parallel with an inhibitory effect on postsynaptic potentials obtained at the The inhibitory activity of adenosine and related compounds on acetylcholine releasefrom enteric nerves is well established (Vizi and Knoll, 1976; Sawynok and Jhamandas, 1976; Hayashi et at., 1978). The locus of action of the nucleosides is clearly a prejunctional interaction with the enteric nerve ending (Gus tafsson et at., 1978) and mediation by a specific nucleoside receptor is supported by the cell surface location of the site of interaction (Okwuasabaet at., 1978) Co., Ltd., Tokyo, Japan). A load of 1.0 g was applied to each prepara tion. Trains of supramaximal rectangular pulses of 1 msec duration were delivered from a Grass model S-5D stimulator (Grass Instruments Co., Quincy, MA) at 0.2 Hz continuously for the duration of each experiment (2â€"3h). Preparations were allowed an equilibration period of 1 h, during which they were washed every 10 mins, before exposure to any drugs. The adenosine analogs R-PIA and NECA were dissolved in 0.5% dimethyl sulfoxide and distilled water, respectively. All drugs were added to the organ bath in aliquots not exceeding 500 zl. Noncumulative log-dose effect curves were obtained for R-PIA and NECA. For other nucleosides, cumulative log dose-effect curves were obtained according to Van Rossum (1963). A period of 20 to 30 mm was allowed between successive determinations of dose-effect curves on a given preparation. Analysis of inhibitory effects of nucleosides. Responseswere expressed as percentage of maximum twitch height. The least-squares method (Snedecor and Cochran, 1967) was used to construct linear regression lines for those data points falling between 20 and 80% of the maximum inhibition of the twitch. Regression lines were con structed for R-PIA and NECA using the pooled data from 8 to 12 preparations and a minimum of five guinea pigs. The concentration resulting in 50% of maximal inhibition (EC@) was determined for these compounds and the corresponding pD2 values The P2 pellet was resuspended to a total volume of 6.0 ml in 0.32 M sucrose and 1.0 ml aliquots were layered onto each of six discontinuous sucrose density gradients consisting of 2.0 ml of 0.8 M sucrose layered On 1 ml of 1.2 M sucrose. After ultracentrifugation at 150,000 x g fo

Canine leishmaniosis in Cyprus due to Leishmania infantum MON 1

During a serological survey in 1996, a total of 601 dogs (group I) distributed all over the government controlled southern area of Cyprus was examined using an enzyme-linked immunosorbent assay (ELISA) for the presence of specific antibodies directed against soluble antigens of promastigote stages of Leishmania infantum. The overall seroprevalence rate in this group was 1.7%. A second group (group II) of dogs was selected from regions where seropositive dogs where determined within the first group. In group II specific anti-Leishmania antibodies could be detected in 30 of 301 dogs investigated (10%). The highest seroprevalence rates were found in the regions of Agios Georgios (26.2%) and Limnatis (12.2%). Ten parasite isolates from ten dogs (six with typical symptoms of canine leishmaniosis and four without symptoms) originating from five locations could be characterised by zymodeme analysis. All ten isolates were identified as L. infantum zymodeme MON 1