Numerical classification and identification of Acinetobacter genomic species

A total of 211 Acinetobacter strains (representing all currently recognized genomic species) were tested for 329 biochemical characters. Overall similarities of all strains were determined for 145 characters by numerical taxonomic techniques, the UPGMA algorithm and the S _SM and the S _J coefficients as measures of similarity. Seven clusters (two or more strains) and three unclustered strains were recovered at a similarity level of 80.0% ( S _SM). At this level a complete correspondence between phenotypic cluster and genomic species was found only for genomic species 12 ( Ac. radioresistens ). At higher similarity levels (84.0% to 84.6% ( S _SM)), however, several subclusters were found, each representing a single genomic species. An exception were the strains belonging to the genetically closely related species of the Acinetobacter calcoaceticus-baumannii complex. These were recovered scattered in several subclusters. The degree of genomic relatedness between some DNA groups correlated with phenotypic similarities, especially for DNA group 8 ( Ac. lwoffii ) and 15 of Tjernberg and Ursing, and for DNA group 4 ( Ac. haemolyticus ) and 6.
For the majority of genomic species, two identification matrices were constructed consisting of 22 and 10 diagnostic characters, respectively. The correct identification rates for the matrices were 98.0% (22 tests) and 90.8% (10 tests) taking a Willcox probability >0.9. For unambiguous identification of some genomic species, however, additional methods (preferably DNA-DNA hybridization or ribotyping) should be used.     

Occurrence and quality of anticoagulant treatment of chronic atrial fibrillation in primary health care in Sweden: a retrospective study on electronic patient records

Background  

Chronic atrial fibrillation is a prevalent cardiac disorder. The literature indicates varying proportions of those treated with anticoagulants, and varying intensity of anticoagulation. Electronic patient records are providing us with clinical data concerning management of anticoagulant treatment in real-life practice that is useful for audits. We aimed to assess warfarin treatment for chronic atrial fibrillation in primary health care with regard to prevalence, incidence, the proportion treated and the quality of anticoagulation control.     

Overexpression of Hsp27 affects the metastatic phenotype of human melanoma cells in vitro

Overexpression of the small heat shock protein Hsp27 has been shown by us to inhibit the in vitro proliferation rate and to delay tumor development of a human melanoma cell line (A375) in nude mice. We hypothesized that Hsp27 may influence the neoplastic phenotype. In the present study Hsp27 transfectants from this cell line were analyzed for various cellular aspects associated with the metastatic process. We found that Hsp27-overexpressing clones exhibited an altered cellular morphology as compared with control transfected cells. The Hsp27-positive cells tended to develop an epithelial-like phenotype growing in clusters and were characterized by a loss of transcytoplasmic stressfibers. In parallel, Hsp27-expressing cells lost the ability to form colonies in soft agar. The invasive potential was studied in vitro by the use of a reconstituted extracellular matrix-coated filter (Matrigel). Compared with controls, Hsp27-overexpressing cells showed decreased cell invasiveness through Matrigel. A correlation between invasion and activation of matrix metalloproteinases (MMPs) has been shown in several cell models. Secretion of MMPs (MMP-2 and MMP-9) was studied by gelatin-substrate zymogram analysis, as well as by a sensitive gelatinase activity assay. The Hsp27-transfected A375 melanoma cell line showed decreased secretion of MMP-2 and MMP-9 as compared with the control transfected cells. Integrins are adhesion receptors and function in cell invasion by mediating cell movement on matrix molecules and by regulating the expression of MMPs. Both fluorescence-activated cell sorter analysis and immunofluorescence analysis revealed a loss of alpha(v)beta3 integrin in Hsp27-transfected cell colonies. Our results demonstrate that Hsp27 overexpression has a profound impact on several parameters regulating the invasive and metastatic potential of melanoma cells in vitro.     

Epoxidation of styrene by human cyt P450 1A2 by thin film electrolysis and peroxide activation compared to solution reactions

Films of human cytochrome P450 1A2 (cyt P450 1A2) and polystyrene sulfonate were constructed on carbon cloth electrodes using layer-by-layer alternate absorption and evaluated for electrochemical- and H(2)O(2)-driven enzyme-catalyzed oxidation of styrene to styrene oxide. At -0.6 V vs. saturated calomel reference electrode in an electrochemical cell, epoxidation of styrene was mediated by initial catalytic reduction of dioxygen to H(2)O(2) which activates the enzyme for the catalytic oxidation. Slightly larger turnover rates for cyt P450 1A2 were found for the electrolytic and H(2)O(2) (10 mM) driven reactions compared to conventional enzymatic reactions using cyt P450s, reductases, and electron donors for cytochromes P450 1A2. Cyt P450(cam) gave comparable turnover rates in film electrolysis and solution reactions. Results demonstrate that cyt P450 1A2 catalyzes styrene epoxidation faster than cyt P450(cam), and suggests the usefulness of this thin-film electrolytic method for relative turnover rate studies of cyt P450s.     

Molecular investigation of the distribution, abundance and diversity of the genus Pseudoalteromonas in marine samples

The genus Pseudoalteromonas has attracted interest because it has frequently been found in association with eukaryotic hosts, and because many Pseudoalteromonas species produce biologically active compounds. One distinct group of Pseudoalteromonas species is the antifouling subgroup containing Pseudoalteromonas tunicata and Ps. ulvae, which both produce extracellular compounds that inhibit growth and colonization by different marine organisms. PCR primers targeting the 16S rRNA gene of the genus Pseudoalteromonas and the antifouling subgroup were developed and applied in this study. Real-time quantitative PCR (qPCR) was applied to determine the relative bacterial abundance of the genus and the antifouling subgroup, and denaturing gradient gel electrophoresis (DGGE) was applied to study the diversity of the genus in 11 different types of marine samples from Danish coastal waters. The detection of Ps. tunicata that contain the antifouling subgroup was achieved through specific PCR amplification of the antibacterial protein gene (alpP). The Pseudoalteromonas species accounted for 1.6% of the total bacterial abundance across all samples. The Pseudoalteromonas diversity on the three unfouled marine organisms Ciona intestinalis, Ulva lactuca and Ulvaria fusca was found to be low, and Ps. tunicata was only detected on these three hosts, which all contain accessible cellulose polymers in their cell walls.     

Effects of Mesolithic hunter-gatherers on local vegetation in a non-uniform glacio-isostatic land uplift area, northern Sweden

The oldest early Mesolithic settlements found so far (i.e. 8600 b.p.) in the interior of northern Sweden, in the province of Norrbotten, have been discovered through the application of a model simulating glacio-isostatic land uplift. The objective of this study was to investigate, through pollen and charred particle analyses, vegetation composition and the influence of man on vegetation and fire pattern in the vicinity of two of these early settlements. Early Holocene vegetation was characterised by Betula, Hippophaë and Salix-species, but no initial impact by hunter-gatherers on vegetation was detected. Subsequently Betula and Pinus became dominant but abrupt changes in the tree layer followed, Poaceae, Hippophaë and Salix-species increased, and Humulus occurred. These changes, which were synchronous with dates of archaeological findings at the settlements, indicated local vegetation changes caused by man. The relationship between these changes and fire was, however, subtle. Eventually Pinus forests became dominant and human impact on vegetation became less apparent. It is concluded that the impact of early Mesolithic hunter-gatherers on vegetation was detectable, but that their effect on fire pattern was difficult to evaluate. Further, we show that the glacio-isostatic land uplift must be considered in the search for Mesolithic settlements in areas previously covered by the Weichselian Ice, and that the process has influenced the biological archives through re-deposition of material.     

Metabolic profiles and genetic diversity of denitrifying communities in activated sludge after addition of methanol or ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

State of degradation in archeological oak from the 17th century vasa ship: substantial strength loss correlates with reduction in (holo)cellulose molecular weight

In 1628, the Swedish warship Vasa capsized on her maiden voyage and sank in the Stockholm harbor. The ship was recovered in 1961 and, after polyethylene glycol (PEG) impregnation, it was displayed in the Vasa museum. Chemical investigations of the Vasa were undertaken in 2000, and extensive holocellulose degradation was reported at numerous locations in the hull. We have now studied the longitudinal tensile strength of Vasa oak as a function of distance from the surface. The PEG-content, wood density, and cellulose microfibril angle were determined. The molar mass distribution of holocellulose was determined as well as the acid and iron content. A good correlation was found between the tensile strength of the Vasa oak and the average molecular weight of the holocellulose, where the load-bearing cellulose microfibril is the critical constituent. The mean tensile strength is reduced by approximately 40%, and the most affected areas show a reduction of up to 80%. A methodology is developed where variations in density, cellulose microfibril angle, and PEG content are taken into account, so that cell wall effects can be evaluated in wood samples with different rate of impregnation and morphologies.     

Identification of flotillin-1 as an interacting protein for antisecretory factor

Antisecretory factor (AF) also named S5a/Rpn10 was originally identified through its capacity to inhibit intestinal hypersecretion and was later shown to be a component in the proteasome complex. AF is also a potent anti-inflammatory agent and can act as a neuromodulator. In this study we used yeast two-hybrid screens, with yeast strain PJ692A transformed with the bait vector pGBKT7 (AF aa 1-105) against yeast strain Y187 pretransformed with human brain or placenta cDNA libraries, to identify AF-binding proteins. Flotillin-1 was identified as a specific interacting factor with AF. Immunohistochemistry showed co-localization of AF and flotillin-1 in nervous tissue. Flotillin-1 is an integral membrane protein and a component of lipid rafts, a membrane specialization involved in transport processes. Intracellular AF may affect secretory processes by regulating the localization of signal proteins to lipid rafts.