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Biomedical subjects

K Johnsen

Publications and source records attributed to K Johnsen.

At least 19 recordsLinked to original sources
PubMed2002

Transformation of trichothecenes in ileal digesta and faeces from pigs.

The capacity of pig gastrointestinal microflora to metabolise the trichothecenes 3-acetyl-deoxynivalenol (3-acDON) and nivalenol (NIV) was investigated. 3-acDON was deacetylated to DON in anaerobic incubations with pig faeces collected at different pig farms. Furthermore, both 3-acDON and NIV were metabolised to the corresponding deepoxy metabolite in these incubates. Five pigs, in which the gastrointestinal microflora lacked the ability to transform 3-acDON and NIV to their corresponding de-epoxidated metabolites, were given low levels of DON in the feed for seven weeks. The gastrointestinal micro-organisms did not acquire the de-epoxidation ability during the seven week long exposure period. At the end of the exposure period, faeces from pigs with a known de-epoxidation ability was spread out in the pens and left for 24 hours. One week after the faeces had been spread out in the pens, the de-epoxidation ability was found in faecal incubations from four out of five experimental pigs. This change in metabolic ability of the intestinal de-epoxidation ability was not accompanied by any detectable changes in the DNA-profiles of the bacterial community composition. The results show that the intestinal de-epoxidation ability is common at pig farms in the Uppsala area, and that the ability may be transferred between pigs in a stock.

Animal Feed↗
PubMed2001

Probing Bose-Einstein condensation of excitons with electromagnetic radiation.

We examine the absorption spectrum of electromagnetic radiation from excitons, where an exciton in the 1s state absorbs a photon and makes a transition to the 2p state. We demonstrate that the absorption spectrum depends strongly on the quantum degeneracy of the exciton gas, and that it will generally manifest many-body effects. Based on our results we propose that absorption of infrared radiation could resolve recent contradictory experimental results on excitons in Cu(2)O.

Journal Article↗
PubMed2001

Single-site mutations induce 3D domain swapping in the B1 domain of protein L from Peptostreptococcus magnus.

BACKGROUND: Thermodynamic and kinetic studies of the Protein L B1 domain (Ppl) suggest a folding pathway in which, during the folding transition, the first beta hairpin is formed while the second beta hairpin and the alpha helix are largely unstructured. The same mutations in the two beta turns have opposite effects on the folding and unfolding rates. Three of the four residues composing the second beta turn in Ppl have consecutive positive phi angles, indicating strain in the second beta turn. RESULTS: We have determined the crystal structures of the beta turn mutants G55A, K54G, and G15A, as well as a core mutant, V49A, in order to investigate how backbone strain affects the overall structure of Ppl. Perturbation of the hydrophobic interactions at the closed interface by the V49A mutation triggered the domain swapping of the C-terminal beta strand that relieved the strain in the second beta turn. Interestingly, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escalated the strain in the second beta turn, and this increased strain shifted the equilibrium toward the domain-swapped dimer. The K54G structure revealed that the increased stability is due to the reduction of strain in the second beta turn, while the G15A structure showed that increased strain alone is insufficient to trigger domain swapping. CONCLUSIONS: Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.

Bacterial Proteins↗
PubMed2001

An altered Pseudomonas diversity is recovered from soil by using nutrient-poor Pseudomonas-selective soil extract media.

We designed five Pseudomonas-selective soil extract NAA media containing the selective properties of trimethoprim and sodium lauroyl sarcosine and 0 to 100% of the amount of Casamino Acids used in the classical Pseudomonas-selective Gould's S1 medium. All of the isolates were confirmed to be Pseudomonas by a Pseudomonas-specific OprF antibody and a Pseudomonas-specific PCR targeting 16S ribosomal DNA. The Pseudomonas isolates were characterized by classical physiological tests, repetitive extragenic palindromic-PCR, Fourier transform infrared spectroscopy, and carbon source utilization patterns. Several of these analyses showed that the amount of Casamino Acids significantly influenced the diversity of the recovered Pseudomonas isolates. Furthermore, the data suggested that specific Pseudomonas subpopulations were represented on the nutrient-poor media. The NAA 1:100 medium, containing ca. 15 mg of organic carbon per liter, consistently gave significantly higher Pseudomonas CFU counts than Gould's S1 when tested on four Danish soils. NAA 1:100 may, therefore, be a better medium than Gould's S1 for enumeration and isolation of Pseudomonas from the low-nutrient soil environment.

Bacterial Typing Techniques↗
PubMed2001

Succession of indigenous Pseudomonas spp. and actinomycetes on barley roots affected by the antagonistic strain Pseudomonas fluorescens DR54 and the fungicide imazalil.

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.

Actinomycetales↗
PubMed2001

Development and application of a most-probable-number-pcr assay to quantify flagellate populations in soil samples.

This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

Animals↗
PubMed2000

Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site.

Phenotypic and genotypic characterization indicated that a group of 29 closely related phenanthrene-degrading bacteria from a coal gasification site in Frederiksberg, Copenhagen, Denmark, belonged to the genus Pseudomonas. The strains were isolated at two sampling occasions 2 years apart. The isolates were phenotypically different from any known species of the genus Pseudomonas and were therefore subject to further identification. Colonies were smooth and pale yellowish and did not produce pigments fluorescent in UV light when grown on King's B agar. Cells were rod-shaped, approximately 0.5-0.8 x 1.5-3.0 microm, and grew at 4 and 30 degrees C, but not 37 degrees C. The bacteria were oxidase- and catalase-positive, accumulated poly-beta-hydroxybutyrate and denitrified, but did not utilize D-xylose. The mean G+C content was 59.6 mol%. Phenotypic data and 16S rDNA sequence data information for Pseudomonas amygdali and Pseudomonas corrugata, and 16S rDNA sequence data for Pseudomonas chlororaphis and Pseudomonas syringae showed close relationships to these strains. However, DNA-DNA hybridization data showed that the isolates belong to a new species, for which the name Pseudomonas frederiksbergensis sp. nov. is proposed. The type strain is JAJ28T (DSM 13022T).

Bacterial Typing Techniques↗
PubMed2000

Crystallization and preliminary X-ray diffraction studies of mutants of B1 IgG-binding domain of protein L from Peptostreptococcus magnus.

The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1. 7, 2.3 and 1.8 A, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites.

Alanine↗
PubMed1999

[Pregnancy and drug abuse].

The antenatal care of pregnant women with drug abuse is very complex and costly both in money and in time. The women often live under different circumstances than non-abusing women, and their personal and social resources are often very small. Collaboration between the hospital and the social system is essential in order to obtain a positive outcome of the pregnancy. Retrospective records of pregnancy, outcome and follow-up on 18 morphine-abusing women referred for delivery at Glostrup hospital in Copenhagen county over the three year period 1992-1994 are reported here. The study show the children are born in relatively good condition, although almost all needed postnatal detoxification, since their mothers was not able to stop their drug abuse during pregnancy. The pregnancy was with few complications because of tight antenatal care for these women. The study also shows a need for defining a common goal for the care of these women and their children and binding political commitments in order to obtain this goal.

Adult↗
PubMed1999

Diversity of Pseudomonas strains isolated with King's B and Gould's S1 agar determined by repetitive extragenic palindromic-polymerase chain reaction, 16S rDNA sequencing and Fourier transform infrared spectroscopy characterisation.

King's B and Gould's S1 agar were compared with regard to the isolation of Pseudomonas from four environmental samples. In all samples, King's B gave the highest number of colony-forming units, and in some environments, there were more fluorescent colony-forming units on King's B as well. However, almost all types grew on Gould's S1, which enabled us to isolate a greater variety of groups than with King's B, fluorescent as well as non-fluorescent members of Pseudomonas. The Pseudomonas isolates were comparatively typed by repetitive extragenic palindromic-polymerase chain reaction and Fourier transform infrared spectroscopy, not previously used for environmental Pseudomonas. The two typing methods were similar in resolution, thus Fourier transform infrared spectroscopy proved fast and reproducible and is a good method for discrimination at subspecies level. Representative strains were identified by partial 16S rDNA sequencing. Thus, we suggest Gould's S1 agar be used for isolation of Pseudomonas because the results are reproducible, specific and give the most diverse recovery and the least work.

Agar↗
PubMed1999

Quantitative selective PCR of 16S ribosomal DNA correlates well with selective agar plating in describing population dynamics of indigenous Pseudomonas spp. in soil hot spots.

We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous Pseudomonas DNA in soil hot spots. The amount of Pseudomonas DNA corresponded to the number of culturable Pseudomonas bacteria on Gould's S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.

Culture Media↗
PubMed1998

Experimental field exposure of brown trout to river water receiving effluent from an integrated newsprint mill.

Effects of effluents from mechanical pulp production on brown trout were studied for 8 weeks at environmentally relevant concentrations. The exposure took place in laboratory-based pools upstream and downstream of the effluent discharge point of an integrated newsprint mill using ground wood/thermomechanical pulp. The mill had no secondary treatment of the wastewater. The pools were supplied with water pumped directly from the river. To determine the relevance of this approach, wild fish were also caught at the respective sites upstream and downstream from the mill. Sublethal effects were assessed using physiological and biochemical parameters including liver histology, hematology, serum biochemistry, and hepatic enzyme assays. Exposure was verified by analyzing water samples, fish bile, and tissues for resin acid concentrations. The downstream experimental fish and captured feral fish displayed responses and changes in physiological parameters similar to those previously observed in laboratory experiments with untreated effluents. The most obvious effects were liver damage and growth inhibition.

Animals↗
PubMed1998

Serosurvey of Toxoplasma gondii in North Atlantic marine mammals by the use of agglutination test employing whole tachyzoites and dithiothreitol.

Serum samples from North Atlantic populations of harp seal, Phoca groenlandica (n = 316), ringed seal, Phoca hispida (n = 48), hooded seal, Cystophora cristata (n = 78), and minke whale, Balaenoptera acutorostrata (n = 202), were tested for Toxoplasma gondii-specific IgG. The modified agglutination test (MAT) was slightly modified to be more user-friendly by replacing the 0.2 M 2-mercaptoethanol with 10 mM dithiothreitol. No positive samples were found at 1:40 dilution.

Agglutination Tests↗
PubMed1997

D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus. I. Kinetic mechanism and pH dependence of kinetic parameters, coenzyme binding and substrate inhibition.

The steady-state kinetics of D-2-hydroxy-4-methylvalerate dehydrogenase have been studied at pH 8.0 by initial velocity, product inhibition, and dead-end inhibition techniques. The mechanism is rapid-equilibrium ordered in the NAD+ plus D-2-hydroxy-4-methylvalerate direction, and steady-state ordered in the other direction. In both cases coenzyme is the first substrate added and both the E-NADH-D-2-hydroxy-4-methylvalerate and E-NAD+-2-oxo-4-methylvalerate give rise to abortive complexes which cause excess substrate inhibition. Steady-state measurements show that the rate-limiting step in both directions at pH 8.0 is between formation of the enzyme-coenzyme-substrate ternary complex and the release of the first product of the reaction. Transient kinetics combined with primary kinetic deuterium isotope effects show that in the NADH-->NAD+ direction there is a slow, rate-limiting rearrangement of the E-NADH-oxoacid complex while hydride transfer is very fast. The release of NAD+ at pH 8.0 is 200-times faster than Kcat (NADH-->NAD+) whereas the release of NADH is only 5-times faster than Kcat (NAD+-->NADH). The pH dependence of NADH binding depends upon the presence of two ionizable residues with a pKa of about 5.9. The pH dependence of kinetic parameters is explained by a third ionizable residue with pKa values 7.2 (in the E-NADH complex) and < or = 6.4 (in the E-NAD+ complex) which may be the proton donor and acceptor for the chemical reaction. At pH 6.5 the mechanism changes in the NADH-->NAD+ direction to be partly limited by the chemical step with a measured primary kinetic isotope effect of 5.7 and partly by an only slightly faster dissociation of NAD+. In addition the inhibition by excess oxo-4-methylvalerate is more pronounced. The mechanism implies that removing the positive charges created by the two groups which control coenzyme affinity could both enhance the catalytic rate at pH 6.5 and diminish excess substrate inhibition to provide an enzyme better suited to the bulk synthesis of D-2-hydroxyacids.

Alcohol Oxidoreductases↗
PubMed1997

D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus. II. Mutagenic analysis of catalytically important residues.

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.

Alcohol Oxidoreductases↗
PubMed1996

Effect of physical activity on serum concentrations of digoxin and digitoxin.

OBJECTIVE: To study the influence of moderate physical activity on serum concentrations of digoxin, digitoxin and albumin. METHODS: Blood samples were drawn from 10 consecutive mobile patients on digoxin and 12 patients on digitoxin therapy before and following a 10-min walking period. Digitalis serum concentrations were determined by radioimmunoassay, and albumin serum concentrations by laser nephelometry. RESULTS: Following physical activity, digoxin serum concentrations dropped immediately to 79% of baseline values and remained significantly decreased for 20 min. Digitoxin concentrations did not change significantly. CONCLUSION: In contrast to digoxin, the effect of physical activity the serum concentration of digitoxin can be disregarded.

Adult↗