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Biomedical subjects

L H Hansen

Publications and source records attributed to L H Hansen.

At least 19 recordsLinked to original sources
PubMed2006

Genetic labelling and application of the isoproturon-mineralizing Sphingomonas sp. strain SRS2 in soil and rhizosphere.

AIMS: To construct a luxAB-labelled Sphingomonas sp. strain SRS2 maintaining the ability to mineralize the herbicide isoproturon and usable for monitoring the survival and distribution of strain SRS2 on plant roots in laboratory systems. METHODS AND RESULTS: We inserted the mini-Tn5-luxAB marker into strain SRS2 using conjugational mating. In the transconjugant mutants luciferase was produced in varying levels. The mutants showed significant differences in their ability to degrade isoproturon. One luxAB-labelled mutant maintained the ability to mineralize isoproturon and was therefore selected for monitoring colonization of barley roots. CONCLUSIONS: We successfully constructed a genetically labelled isoproturon-mineralizing-strain SRS2 and demonstrated its ability to survive in soil and its colonization of rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: The construction of a luxAB-labelled strain SRS2 maintaining the degradative ability, provides a powerful tool for ecological studies serving as the basis for evaluating SRS2 as a bioremediation agent.

Biodegradation, Environmental↗
PubMed2003

Presence of N-acyl homoserine lactones in soil detected by a whole-cell biosensor and flow cytometry.

Quorum sensing enables bacteria to regulate expression of certain genes according to population density. N-acyl homoserine lactone (AHL)-based quorum sensing is known to be widespread among gram-negative bacteria. Several bacterial whole-cell biosensors for AHL detection have been developed and some were used in in situ studies of AHL production. From these studies our knowledge of the significance of quorum sensing in various environments has been improved. However, very little is known about production of AHLs in soil environments. In the present study, an approach for detecting AHL production in bulk soil was developed. A whole-cell biosensor based on the regulatory region of the lux-operon from Vibrio fischeri fused to gfp was constructed, resulting in a luxR-PluxI-gfpmut3*-fusion in the high copy plasmid, pAHL-GFP. Escherichia coli MC4100 harboring pAHL-GFP responded to the AHL-compound N-octanoyl homoserine lactone (OHL) by expressing green fluorescence. In situ application of E. coli MC4100/pAHL-GFP was tested by adding OHL in different concentrations to sterile soil microcosms. E. coli MC4100/pAHL-GFP were incubated in the soil microcosms and extracted by an improved Nycodenz-extraction method optimized for flow cytometry. The presence of induced cells was then verified by single-cell analysis by flow cytometry. OHL concentrations between 0.5 and 50 nmol per g soil were detected. When introducing the AHL-producing Serratia liquefaciens to soil microcosms, expression of green fluorescent protein was induced in E. coli MC4100/pAHL-GFP. Thereby, the ability of this strain to detect excretion of AHLs by S. liquefaciens in sterile soil was shown. The use of an improved extraction method and a whole-cell biosensor combined with flow cytometry analysis proved to be promising tools in future studies of AHL production by microbial populations in soil environments.

4-Butyrolactone↗
PubMed2001

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase.

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.

Anti-Bacterial Agents↗
PubMed2001

Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry.

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

Biosensing Techniques↗
PubMed2000

Versatile biosensor vectors for detection and quantification of mercury.

Three different whole cell biosensor constructs were made by fusing the mercury inducible promoter, P(mer), and its regulatory gene, merR, from transposon Tn21 with reporter genes luxCDABE, lacZYA, or gfp. In Escherichia coli these biosensor constructs responded to low levels of mercury by producing light, beta-galactosidase or green fluorescent protein, respectively. Since the responses were quantitative, the constructs were used to quantify bioavailable mercury in different environments. The constructs were cloned into mini-Tn5 delivery vectors, thus enabling the transfer of the mer-lux, mer-lac or mer-gfp cassettes to a variety of Gram-negative bacteria. The mer-lux cassette was transferred to a Pseudomonas putida strain, which was used to quantify water-extractable mercury in contaminated soil.

Bacterial Proteins↗
PubMed2000

Detection and quantification of tetracyclines by whole cell biosensors.

Three different mini-Tn5 plasmids, containing a tetracycline-inducible promoter, Ptet and a regulatory gene, tetR, in operon fusions with a reporter gene system (lacZYA, luxCDABE or gfp), were constructed. These biosensor constructs responded to low levels of tetracyclines by producing beta-galactosidase, light or green fluorescent protein. They did so in a quantitative manner, thus enabling the quantification of tetracyclines in the immediate surroundings of the biosensor organism. All three constructs were transferred successfully to different gram-negative bacteria by conjugation. An Escherichia coli strain containing the Ptet-lac construct was used to determine oxytetracycline in milk as a demonstration of the application of these biosensors.

Animals↗
PubMed2000

Macrolide-ketolide inhibition of MLS-resistant ribosomes is improved by alternative drug interaction with domain II of 23S rRNA.

The macrolide antibiotic erythromycin and its 6-O-methyl derivative (clarithromycin) bind to bacterial ribosomes primarily through interactions with nucleotides in domains II and V of 23S rRNA. The domain II interaction occurs between nucleotide A752 and the macrolide 3-cladinose moiety. Removal of the cladinose, and substitution of a 3-keto group (forming the ketolide RU 56006), results in loss of the A752 interaction and an approximately 100-fold drop in drug binding affinity. Within domain V, the key determinant of drug binding is nucleotide A2058 and substitution of G at this position is the major cause of drug resistance in some clinical pathogens. The 2058G mutation disrupts the drug-domain V contact and leads to a further > 25 000-fold decrease in the binding of RU 56006. Drug binding to resistant ribosomes can be improved over 3000-fold by forming an alternative and more effective contact to A752 via alkyl-aryl groups linked to a carbamate at the drug 11/12 position (in the ketolide antibiotics HMR 3647 and HMR 3004). The data indicate that simultaneous drug interactions with domains II and V strengthen binding and that the domain II contact is of particular importance to achieve binding to the ribosomes of resistant pathogens in which the domain V interaction is perturbed.

Anti-Bacterial Agents↗
PubMed1999

Nickel-induced activation of T cells in individuals with negative patch test to nickel sulphate.

Contact hypersensitivity to nickel is the most common form of allergic contact dermatitis. To gain insight into the induction of this frequent disease, T cell reactivity towards nickel was investigated in "nonallergic" individuals defined as those with no skin manifestations and a negative patch test towards NiSO4. Surprisingly, we found that nickel induced proliferation of peripheral blood mononuclear cells (PBMC) from 16 of 18 adult individuals tested. This activation was specific, and no stimulation of PBMC was observed using control stimulants at equimolar concentrations. Furthermore, the NiSO4-induced activation required the presence of professional antigen-presenting cells. To describe the functional capacity of the nickel-inducible T cells, cytokine release was investigated in both nickel-allergic and nonallergic individuals. The T cells from both groups released interferon-gamma but no interleukin-4 upon stimulation with nickel, suggesting that the functional capacities of these cell populations were similar in nickel-allergic and nonallergic individuals. Thus, at this level, no qualitative differences could be demonstrated between T cells obtained from nickel-allergic and nonallergic individuals.

Adult↗
PubMed1999

Core sequence in the RNA motif recognized by the ErmE methyltransferase revealed by relaxing the fidelity of the enzyme for its target.

Under physiological conditions, the ErmE methyltransferase specifically modifies a single adenosine within ribosomal RNA (rRNA), and thereby confers resistance to multiple antibiotics. The adenosine (A2058 in Escherichia coli 23S rRNA) lies within a highly conserved structure, and is methylated efficiently, and with equally high fidelity, in rRNAs from phylogenetically diverse bacteria. However, the fidelity of ErmE is reduced when magnesium is removed, and over twenty new sites of ErmE methylation appear in E. coli 16S and 23S rRNAs. These sites show widely different degrees of reactivity to ErmE. The canonical A2058 site is largely unaffected by magnesium depletion and remains the most reactive site in the rRNA. This suggests that methylation at the new sites results from changes in the RNA substrate rather than the methyltransferase. Chemical probing confirms that the rRNA structure opens upon magnesium depletion, exposing potential new interaction sites to the enzyme. The new ErmE sites show homology with the canonical A2058 site, and have the consensus sequence aNNNcgGAHAg (ErmE methylation occurs exclusively at adenosines (underlined); these are preceded by a guanosine, equivalent to G2057; there is a high preference for the adenosine equivalent to A2060; H is any nucleotide except G; N is any nucleotide; and there are slight preferences for the nucleotides shown in lower case). This consensus is believed to represent the core of the motif that Erm methyltransferases recognize at their canonical A2058 site. The data also reveal constraints on the higher order structure of the motif that affect methyltransferase recognition.

Adenine↗
PubMed1999

Nickel-induced proliferation of both memory and naive T cells in patch test-negative individuals.

Lymphocyte transformation test has often been used as an in vitro test for nickel allergy. We have previously demonstrated the presence of nickel-reactive T cells in individuals with no history of allergic disease and with a negative patch test towards NiSO4. In this study, we show that this proliferative response was mainly confined to T cells within the CD4+ subset. In contrast to conventional recall antigens such as tetanus toxoid, in vitro stimulation using NiSO4 activated both FACS-purified CD4+CD45RA+ 'naive' and CD4+CD45RO+ 'memory' T cells. To determine which cell population reacted with nickel to induce T cell activation, peripheral blood mononuclear cells were separated into macrophages and non-adherent, HLA-DR-depleted T cells. We found that preincubation of monocytes/macrophages but not T cells with NiSO4 resulted in subsequent T cell proliferation. This result demonstrated that nickel did not exhibit any direct effect on the T cell. Furthermore, the NiSO4-induced T cell proliferation could be blocked by antibodies towards MHC class II (HLA-DR) molecules. Our results substantiate the concept that individuals with a negative patch test towards NiSO4 contain in their peripheral blood T cells capable of recognizing nickel or nickel-modified peptides. In contrast to conventional recall antigens, both memory and naive T cells were activated. Thus, when compared with data obtained from nickel-allergic individuals, this study shows a comparable nickel-inducible T cell activation in non-allergic individuals.

Adult↗
PubMed1999

The macrolide-ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA.

The macrolide antibiotic erythromycin interacts with bacterial 23S ribosomal RNA (rRNA) making contacts that are limited to hairpin 35 in domain II of the rRNA and to the peptidyl transferase loop in domain V. These two regions are probably folded close together in the 23S rRNA tertiary structure and form a binding pocket for macrolides and other drug types. Erythromycin has been derivatized by replacing the L-cladinose moiety at position 3 by a keto group (forming the ketolide antibiotics) and by an alkyl-aryl extension at positions 11/12 of the lactone ring. All the drugs footprint identically within the peptidyl transferase loop, giving protection against chemical modification at A2058, A2059 and G2505, and enhancing the accessibility of A2062. However, the ketolide derivatives bind to ribosomes with widely varying affinities compared with erythromycin. This variation correlates with differences in the hairpin 35 footprints. Erythromycin enhances the modification at position A752. Removal of cladinose lowers drug binding 70-fold, with concomitant loss of the A752 footprint. However, the 11/12 extension strengthens binding 10-fold, and position A752 becomes protected. These findings indicate how drug derivatization can improve the inhibition of bacteria that have macrolide resistance conferred by changes in the peptidyl transferase loop.

Anti-Bacterial Agents↗
PubMed1998

ErmE methyltransferase recognition elements in RNA substrates.

Dimethylation by Erm methyltransferases at the N-6 position of adenine 2058 (A2058, Escherichia coli numbering) in domain V of bacterial 23 S rRNA confers resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics. The ErmE methyltransferase from Saccharopolyspora erythraea methylates a 625 nucleotide transcript of domain V as efficiently as it methylates intact 23 S rRNA. By progressively truncating domain V, the motif required for specific recognition by the enzyme has been localized to a helix and single-stranded region adjacent to A2058. The smallest RNA transcript that shows methyl-accepting activity is a 27-nucleotide stem-loop, corresponding to the 23 S rRNA sequences 2048 to 2063 and 2610 to 2620 (helix 73), with A2058 situated within the hairpin loop. Methylation of A2058 in the truncated RNAs is optimal in the absence of magnesium, and the efficiency of methylation is halved by the presence of 2 to 3 mM magnesium. Magnesium serves to stabilize a conformation in the truncated RNA that prevents efficient methylation. This contrasts to the intact domain V RNA, where 2 mM magnesium ions support a conformation at A2058 that is most readily recognized by ErmE. Methylation of domain V RNA is generally far less susceptible to ionic conditions than the truncated RNAs. The effects of monovalent cations on the methylation of truncated transcripts suggest that RNA structures outside helix 73 support the ErmE interaction. However, interaction with these structures is not essential for specific ErmE recognition of A2058.

Ammonium Chloride↗
PubMed1998

[Amebiasis--a differential diagnosis from inflammatory bowel disease].

Entamoeba histolytica is a very uncommon etiology of travelers diarrhoea, but may be an important differential diagnosis to inflammatory bowel disease. A misdiagnosis of amoebic colitis as inflammatory bowel disease followed by inadvertent treatment with corticosteroids may be fatal. Current techniques for the diagnosis of amoebiasis include stool studies, endoscopic biopsy and serology. Demonstration of haematophagous trophozoites in feces or in tissue biopsy is the definitive method for diagnosis. Unfortunately both methods have many drawbacks. If the specimens are negative, serology can support the diagnosis especially in a non-endemic area where a positive test is highly predictive of current disease.

Aged↗
PubMed1998

The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens.

The role of the Escherichia coli lacY gene product (the lactose permease) in the induction of isopropyl-beta-D-thiogalactopyranoside (IPTG) inducible promoters was studied in E. coli and P. fluorescens. This was done by comparing strains containing a lacIPOZYA chromosomal insert with newly constructed strains containing inserts without the lacY gene (lacIPOZ). The lactose operon inserts were introduced as single-copy chromosomal inserts to eliminate differences in expression caused by differences in copy number. Comparison between the two types of inserts showed that the lactose permease was essential to allow growth on lactose by both bacteria and that the lactose permease plays an important role in transporting the inducer IPTG across the membrane of P. fluorescens. The use of a functional lactose permease allows expression of beta-galactosidase to increase more than fivefold from a wild-type lac promoter in P. fluorescens SS1001. We suggest that an increase in the rate of protein synthesis from lac-type promoters could be enhanced if an active lactose permease is present as well.

Escherichia coli↗
PubMed1998

Glucagon-mediated Ca2+ signaling in BHK cells expressing cloned human glucagon receptors.

From video imaging of fura 2-loaded baby hamster kidney (BHK) cells stably expressing the cloned human glucagon receptor, we found the Ca2+ response to glucagon to be specific, dose dependent, synchronous, sensitive to pertussis toxin, and independent of Ca2+ influx. Forskolin did not elicit a Ca2+ response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3',5'-cyclic monophosphothioate, resulted in a reduced glucagon-mediated Ca2+ response as well as Ca2+ oscillations. The specific phospholipase C inhibitor U-73122 abolished the Ca2+ response to glucagon, and a modest twofold increase in inositol trisphosphate (IP3) production could be observed after stimulation with glucagon. In BHK cells coexpressing glucagon and muscarinic (M1) acetylcholine receptors, carbachol blocked the rise in intracellular free Ca2+ concentrations in response to glucagon, whereas glucagon did not affect the carbachol-induced increase in Ca2+. Furthermore, carbachol, but not glucagon, could block thapsigargin-activated increases in intracellular free Ca2+ concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation of phospholipase C and the release of Ca2+ from IP3-sensitive intracellular Ca2+ stores. Finally, we provide evidence to suggest that cAMP potentiates the IP3-mediated effects on intracellular Ca2+ handling.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Characterization of the inhibitory effect of growth hormone on primary preadipocyte differentiation.

GH exerts adipogenic activity in several preadipocyte cell lines, whereas in primary rat preadipocytes, GH has an antiadipogenic activity. To better understand the molecular mechanism involved in adipocyte differentiation, the expression of adipocyte-specific genes was analyzed in differentiating preadipocytes in response to GH. We found that the expression of both adipocyte determination and differentiation factor 1 (ADD1) and peroxisome proliferator activated receptor gamma(PPARgamma) was induced in preadipocytes during differentiation. In the presence of GH, which markedly inhibited triglyceride accumulation, no reduction in the expression level of ADD1 was observed in response to GH, whereas there was a 50% reduction in the expression of PPARgamma. The DNA binding activity of the PPARgamma/retinoid X receptor-alpha(RXRalpha) to the ARE7 element from the aP2 gene was also reduced by approximately 50% in response to GH. GH inhibited the expression of late markers of adipocyte differentiation, fatty acid synthase, aP2, and hormone-sensitive lipase by 70-80%. The antiadipogenic effect of GH was not affected by the mitogen-activated protein (MAP) kinase/ extracellular-regulated protein (ERK) kinase inhibitor PD 98059, indicating that the mitogen-activated protein kinase pathway was not involved in GH inhibition of preadipocyte differentiation. The expression of preadipocyte factor-1/fetal antigen 1 was decreased during differentiation, and GH treatment prevented this down-regulation of Pref1/FA1. A possible role for Pref-1/FA1 in mediating the antiadipogenic effect of GH was indicated by the observation that FA1 inhibited differentiation as effectively as GH. These data suggest that GH exerts its inhibitory activity in adipocyte differentiation at a step after the induction of ADD1 but before the induction of genes required for terminal differentiation.

Adipocytes↗
PubMed1997

Chromosomal insertion of the entire Escherichia coli lactose operon, into two strains of Pseudomonas, using a modified mini-Tn5 delivery system.

A 12-kb PstI fragment including the entire E. coli lactose operon (lacIPOZYA) was inserted in one copy into the chromosome of Pseudomonas putida, Pseudomonas fluorescens and an E. coli strain with lac- phenotype. This was made possible by improvements of an already existing mini-Tn5 transposon delivery system (de Lorenzo et al., 1990; Herrero et al., 1990), which integrates cloned DNA fragments at random sites on the chromosome of the recipient bacteria in single copies. This has resulted in: (a) the making of two useful low copy-number cloning vectors both with extensive multi-cloning regions flanked by NotI sites needed in the mini-Tn5 delivery system; (b) the generation of E. coli nonlysogenic strains expressing the pi protein thus being capable of maintaining and delivering R6K-based mini-Tn5 vectors to other E. coli strains; (c) the successful insertion of the E. coli lactose operon into the P. fluorescens chromosome giving P. fluorescens the ability to grow on lactose; (d) evidence from Southern blotting that contradicts the assumption that the mini-Tn5 delivery system always creates one-copy inserts. These improvements allow insertion of large DNA fragments encoding highly expressed proteins into the chromosome of a large variety of Gram-negative bacteria including E. coli.

Chromosomes, Bacterial↗