Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase

Long-term cultivation of the Pseudomonas putida multiplasmid strain EST1020 on phenol resulted in the formation of individual PHE plasmids determining phenol degradation. Four types of PHE plasmids, pEST1024, pEST1026, pEST1028, and pEST1029, are characterized. They all contain a transferrable replicon similar to pWWO-8 with a partly duplicated DNA sequence of the 17-kb transposable element of this plasmid and include various amounts of DNA that carry genes encoding phenol degradation (phe genes). We cloned the genes determining phenol monooxygenase and catechol 1,2-dioxygenase from the Pseudomonas sp. parent strain plasmid DNA into the broad host range vector pAYC32 and studied the expression of the cloned DNA. The formation of a new hybrid metabolic plasmid, pEST1354, was demonstrated in P. putida PaW85 as the result of transposition of the 17-kb genetic element from the chromosome of PaW85 into the plasmid carrying cloned phe genes. The target site for the 17-kb transposon was localized in the vector DNA, just near the cloning site. In subcloning experiments we found two regions in the 17-kb DNA stretch that are involved in the expression of the cloned phe genes.     

Contribution of P2X4 receptor in pain associated with rheumatoid arthritis: a review

Purinergic Signalling - Pain is the most common symptom reported by patients with rheumatoid arthritis (RA) even after the resolution of chronic joint inflammation. It is believed that...     

Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil,shale oil or diesel fuel

The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea.     

Characterization of M2 muscarinic receptor activation of different G protein subtypes

The M2 muscarinic acetylcholine receptor (mAChR) expressed in insect cells (Spodoptera frugiperda, Sf9) using the baculovirus system formed active functional complexes with coexpressed Gi as well as with Go proteins, while no complexes could be detected with internal G proteins. Comparison of the abilities of different muscarinic agonists and partial agonists to increase [35S]GTPgammaS binding revealed no significant differences between M2/Gi and M2/Go complexes neither with respect to affinities nor efficacies of the ligands studied. Coexpression with either G protein caused constitutive activity of the receptor amounting up to 66% of stimulable [35S]GTPgammaS binding. Muscarinic antagonists, like atropine, scopolamine and N-methylscopolamine, behaved as inverse antagonists with potencies in good agreement with their binding affinities to the receptor. The results implicate that the functional reconstitution of M2 muscarinic receptor with either Gi or Go proteins in insect cells provides a valuable tool for screening of potencies as well as efficacies of agonists, partial agonists and inverse agonists at this receptor.     

Addition of biological functionality to poly(epsilon-caprolactone) films

Biodegradable polyesters such as poly(epsilon-caprolactone) (PCL) have a number of biomedical applications; however, their usage is often limited by a lack of biological functionality. In this paper, a PCL-based polymer containing pendent groups activated by 4-nitrophenyl chloroformate (NPC) and reactive toward primary amines has been cast into thin films. The reactivity of the films toward poly(l-lysine) and the cell adhesion peptide, GRGDS, was assessed, and their cell adhesive capabilities were characterized. ATR-FTIR analysis found that NPC functional groups were present on the surface of the cast film, and the synthesis, conjugation, and visualization of a fluorescent molecule on these films further demonstrated the success of this functionalization methodology. The immersion of these films into a solution of either poly(l-lysine) (PLL) or GRGDS in PBS (pH 7.4) and subsequent 3T3 fibroblast adhesion studies demonstrated significant improvement in cell adhesion and spreading over films cast from unmodified PCL. This investigation has shown that this novel NPC-containing polymer can be utilized in many applications where increased cellular adhesion is required, or the coupling of specific molecules to polymer surfaces is of interest.     

Dynamics of gaseous nitrogen and carbon fluxes in riparian alder forests

We studied greenhouse gas (GHG) fluxes in two differently loaded riparian Alnus incana-dominated forests in agricultural landscapes of southern Estonia: a 33-year-old stand in Porijõgi, in which the uphill agricultural activities had been abandoned since the middle of the 1990s, and a 50-year-old stand in Viiratsi, which still receives polluted lateral flow from uphill fields fertilized with pig slurry. In Porijõgi, closed-chamber based sampling lasted from October 2001 to October 2009, whereas in Viiratsi the sampling period was from November 2003 to October 2009. Both temporal and spatial variations in all GHG gas fluxes were remarkable. Local differences in GHG fluxes between micro-sites (“Edge”, “Dry” and “Wet” in Porijõgi, and “Wet”, “Slope” and “Dry” in Viiratsi) were sometimes greater than those between sites. Median values of GHG fluxes from both sites over the whole study period and all microsites did not differ significantly, being 45 and 42 mg CO₂-C m⁻² h⁻¹, 8 and 0.5 μg CH₄-C m⁻² h⁻¹, 1.0 and 2.1 mg N₂-N m⁻² h⁻¹, and 5 and 9 μg N₂O-N m⁻² h⁻¹, in Porijõgi and Viiratsi, respectively. The N₂:N₂O ratio in Viiratsi (40-1200) was lower than in Porijõgi (10-7600). The median values-based estimation of the Global Warming Potential of CH₄ and N₂O was 19 and 185 kg CO₂ equivalents (eq) ha⁻¹ yr⁻¹ in Porijõgi and −14 and 336 kg CO₂ eq ha⁻¹ yr⁻¹ in Viiratsi, respectively. A significant Spearman rank correlation was found between the mean monthly air temperature and CO₂, CH₄ and N₂ fluxes in Porijõgi, and N₂O flux in Viiratsi, and between the monthly precipitation and CH₄ fluxes in both study sites. Higher groundwater level significantly increases CH₄ emission and decreases CO₂ and N₂O emission, whereas higher soil temperature significantly increases N₂O, CH₄ and N₂ emission values. In Porijõgi, GHG emissions did not display any discernable trend, whereas in Viiratsi a significant increase in CO₂, N₂, and N₂O emissions has been found. This may be a result of the age of the grey alder stand, but may also be caused by the long-term nutrient load of this riparian alder stand, which indicates a need for the management of similar heavily loaded riparian alder stands.     

Kinetic and functional properties of [3H]ZM241385, a high affinity antagonist for adenosine A2A receptors

We have characterized the binding of [2-(3)H]-4-(2-[7-Amino-2-(2-furyl)-[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol ([(3)H]ZM241385) to adenosine A(2A) receptors in membranes of rat striatum and transfected CHO cells. Saturation experiments showed that [(3)H]ZM241385 binds to a single class of binding sites with high affinity (K(d) = 0.23 nM and 0.14 nM in CHO cell and striatal membranes, respectively). The membranes of CHO cells required pretreatment with adenosine deaminase (ADA) to achieve high-affinity binding, while ADA had no influence on the ligand binding properties in striatal membranes. The binding of [(3)H]ZM241385 was fast and reversible, achieving equilibrium within 20 minutes at all radioligand concentrations. The kinetic analysis of the [(3)H]ZM241385 interaction with A(2A) receptors indicated that the reaction had at least two subsequent steps. The first step corresponds to a fast equilibrium, which also determines the antagonist potency to competitively inhibit CGS21680-induced accumulation of cAMP (first equilibrium constant K(A) = 6.6 nM). The second step corresponds to a slow process of conformational isomerization (equilibrium constant K(i) = 0.03). The combination of the two steps gives the dissociation constant K(d) = 0.20 nM based on the kinetic data, which is in good agreement with the directly measured value. The data obtained shed light on the mechanism of the [(3)H]ZM241385 interaction with adenosine A(2A) receptors from different sources in vitro. The isomerization step of the A(2A) antagonist radioligand binding has to be taken into account for the interpretation of the binding parameters obtained from the various competition assays and explain the discrepancy between antagonist affinity in saturation experiments versus its potency in functional assays.     

General Description of Partly Meromictic Hypertrophic Lake Verevi,its Ecological Status,Changes during the Past Eight Decades,and Restoration Problems

The present study describes generally the ecosystem of Lake Verevi while more detailed approaches are presented in the same issue. The main task of the article is to estimate long-term changes and find the best method for the restoration of good ecological status. Lake Verevi (surface 12.6 ha, mean depth 3.6 m, maximum depth 11 m, drainage area 1.1 km², water exchange 0.63-times per year) is a hypertrophic hard-water lake located in town Elva (6400 inhabitants). Long-term complex limnological investigations have taken place since 1929. The lake has been contaminated by irregular discharge of urban wastewaters from oxidation ponds since 1978, flood from streets, and infiltrated waters from the surrounding farms. The so-called spring meromixis occurred due to extremely warm springs in recent years. The index value of buffer capacity of Lake Verevi calculated from natural conditions is on the medium level. Water properties were analysed according to the requirements of the EU Water Framework Directive. According to the classification, water quality as a long-term average of surface layers is moderate-good, but the water quality of bottom layers is bad. Values in deeper layers usually exceed 20–30 times the calculated reference values by Vighi and Chiaudani’s model. Naturally, at the beginning of the 20th century the limnological type of the lake was moderately eutrophic. During the 1980s and 1990s the ecosystem was out of balance by abiotic characteristics as well as by plankton indicators. Rapid fluctuations of species composition and abundance can be found in recent years. Seasonal variations are considerable and species composition differs remarkably also in the water column. The dominating macrophyte species vary from year to year. Since the annual amount of precipitation from the atmosphere onto the lake surface is several times higher, the impact of swimmers could be considered irrelevant. Some restoration methods were discussed. The first step, stopping external pollution, was completed by damming the inlet. Drainage (siphoning) of the hypolimnetic water is discussed. Secondary pollution occurs because Fe:P values are below the threshold. The authors propose to use phosphorus precipitation and hypolimnetic aeration instead of siphoning.