Biomedical subjects

Anna Karlsson

Publications and source records attributed to Anna Karlsson.

At least 37 records · Page 2Linked to original sources

PubMed2005

Feasibility of a computerized alcohol screening and personalized written advice in the ED: opportunities and obstacles.

This study evaluates the feasibility of a computerized alcohol screening and intervention in patients seeking care at an emergency department. The aim of the study was to explore prevailing attitudes among nursing staff to alcohol prevention in general and the computerized screening concept before the introduction of the computerized screening procedure. Interviews were performed with six nursing staff members and a written questionnaire applied to all staff participating in training before the implementation of the concept. Observation was done in order to evaluate how the screening was performed and implemented in order to gain more knowledge about possible obstacles to alcohol screening at an emergency department. The project concept of computerized screening and low staff involvement was found to be a feasible way to overcome organizational barriers such as time limitation but there were still important attitudinal barriers among staff; thus a great disparity in practices between different staff members was observed. The results suggest that it is possible, given the time limitation, to include a computerized screening and intervention in the routines of an emergency department, when the staff regard prevention as part of their responsibilities. The findings provide evidence about the acceptability by nursing staff and feasibility of a computerized screening and personalized print out as feed back to the patients.

Alcohol-Related Disorders↗

PubMed2005

Acceptability of a computerized alcohol screening and advice routine in an emergency department setting--a patient perspective.

Disseminating screening and brief alcohol intervention in routine emergency department (ED) care seems to be a demanding challenge due to the complexity of the emergency setting. Further empirical realistic intervention models need to be developed that are appropriate for staff and patients before such models are tested in large scale controlled trials. This study explores patients' acceptance of a computerized opportunistic alcohol screening, followed by a personalized printout as feedback, at an emergency department performed by ordinary emergency department staff. In all, 44 patients were interviewed after performing the computerized screening test. The majority of patients were positive about answering the questions about alcohol habits, and 95% found it easy to use the touch screen and understand the instructions, even those with little computer experience. Regarding the personalized printout of advice, 76% were positive to this kind of feedback and 93% said they would read the printout. Most patients, 74%, preferred the computerized printout instead of a more personalized feedback from staff members. Whether the computerized advice is sufficient for a change in alcohol habits among excessive drinkers is still to be shown in controlled trials, but the concept could be one means of making preventive measures more feasible in a real-world ED setting. This might provide a basis for dissemination and integration of more extensive interventions in the long term.

Alcohol Drinking↗

PubMed2005

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster.

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.

Animals↗

PubMed2005

Interleukin-8-derived peptide has antibacterial activity.

Chemokines are inflammatory mediators with effects on diverse processes associated with the immune response. Some of the proteins belonging to the CXC chemokine subfamily, one of four groups in the family, possess inherent antibacterial activity against a wide range of bacteria. The CXC chemokine interleukin-8 (IL-8) has not been ascribed any direct antibacterial activity, but the fact that several of the amino acids in the carboxy-terminal part of the protein are identical or similar to those in a bactericidal cecropin-like peptide [Hp(2-20)] from Helicobacter pylori suggests that processing of the cytokine might generate peptide fragments with antibacterial properties. Synthetic peptides representing the carboxy-terminal part of IL-8 were investigated for antibacterial activities. These fragments possessed an antibacterial activity absent in the full-length IL-8. The antibacterial effects were reduced at increasing salt concentrations whereas the activity was increased when the pH was lowered. The IL-8-derived peptide shared structural similarity with and was also functionally additive to the Hp(2-20) peptide. The IL-8-derived peptide lacked the proinflammatory effects of the full-length protein. We also showed that acid hydrolysis of IL-8 generated a major peptide fragment corresponding to the antibacterial carboxyl terminus of the protein. The results presented are of special interest when put in the context of the suggested importance of antimicrobial peptides for microbial colonization of the gastric mucosa.

Amino Acid Sequence↗

PubMed2004

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide.

BACKGROUND: The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites. RESULTS: We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton. CONCLUSIONS: This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.

Cytoplasmic Granules↗

PubMed2004

Synthesis and evaluation of thymine-derived carboxamides against mitochondrial thymidine kinase (TK-2) and related enzymes.

Based on the structure of our previously identified mitochondrial thymidine kinase (TK-2) inhibitors, three series of thymine-derived carboxamides have been synthesized and tested against TK-2 and related enzymes. The methodology employed has been a solution-phase parallel synthesis based on the coupling of three thymine-derived acids [4-(thymin-1-yl)butyric acid (I), [4-(thymin-1-yl)-butyrylamino]acetic acid (II) and 6-(thymin-1-yl)hexanoic acid (III)] with different commercially available primary amines that carry cyano and/or phenyl groups. The couplings were performed in good yields (from 60% to 90%), with the exception of those that incorporate the highly crowded triphenylmethylamine (e). From the new synthesized compounds, the N-trityl-6-(thymin-1-yl)hexanamide (IIIe) was the most active TK-2 inhibitor (IC(50)=19+/-2microM).

Amino Acids↗

PubMed2004

Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe.

Human neutrophils are activated by the beta-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS. Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge. Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3. Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV). In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase. The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid. Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors. Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3. Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.

Cell Degranulation↗

PubMed2004

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils.

BACKGROUND: Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions. RESULTS: Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state. CONCLUSIONS: The TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.

Cyclosporine↗

PubMed2004

Occurrence and abatement of volatile sulfur compounds during biogas production.

Volatile sulfur compounds (VSCs) in biogas originating from a biogas production plant and from a municipal sewage water treatment plant were identified. Samples were taken at various stages of the biogas-producing process, including upgrading the gas to vehicle-fuel quality. Solid-phase microextraction was used for preconcentration of the VSCs, which were subsequently analyzed using gas chromatography in combination with mass spectrometry. Other volatile organic compounds present also were identified. The most commonly occurring VSCs in the biogas were hydrogen sulfide, carbonyl sulfide, methanethiol, dimethyl sulfide, and dimethyl disulfide, and hydrogen sulfide was not always the most abundant sulfur (S) compound. Besides VSCs, oxygenated organic compounds were commonly present (e.g., ketones, alcohols, and esters). The effect of adding iron chloride to the biogas reactor on the occurrence of VSCs also was investigated. It was found that additions of 500-g/m3 substrate gave an optimal removal of VSCs. Also, the use of a prefermentation step could reduce the amount of VSCs formed in the biogas process. Moreover, in the carbon dioxide scrubber used for upgrading the gas, VSCs were removed efficiently, leaving traces (ppbv levels). The scrubber also removed other organic compounds.

Air Pollutants↗

PubMed2004

The mechanism for activation of the neutrophil NADPH-oxidase by the peptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs from that for interleukin-8.

Neutrophil chemotaxis has been shown to be regulated by two different signalling pathways that allow strong chemoattractants, such as bacterial-derived formylated peptides, to dominate over endogenous attractants, such as interleukin-8 (IL-8). Here we show that triggering of the formyl peptide receptor (FPR) with f-Met-Leu-Phe (fMLF) substantially reduced the neutrophil superoxide production induced by activation of the CXC receptors with IL-8. When the order of agonists was reversed, the cells were primed in their response to fMLF, suggesting that the signalling hierarchy between strong, so-called end-type (i.e. fMLF) and weak or intermediate-type (i.e. IL-8) chemoattractants, is also operating during activation of the NADPH-oxidase. The same result was obtained when fMLF was replaced with the hexapeptide, WKYMVM, specific for the formyl peptide-like receptor 1 (FPRL1). There were additional differences between the agonist receptor pairs fMLF/FPR, WKYMVM/FPRL1 and IL-8/CXCR. In contrast to FPR and FPRL1, no reserve pool of CXCR was present in subcellular granules and it was impossible to prime the oxidative response transduced through CXCR by the addition of priming agents such as tumour necrosis factor-alpha and platelet-activating factor. Moreover, the cytoskeleton-disrupting substance, cytochalasin B, had no effect either on IL-8-triggered oxidase activation or on CXCR reactivation. A pertussis toxin-sensitive G-protein is involved in signalling mediated through both FPR and CXCR, and the signalling cascades include a transient intracellular calcium increase, as well as downstream p38 MAPK and phosphoinositide 3-kinase activation. The data presented in this study provide support for two different signalling pathways to the neutrophil NADPH-oxidase, used by ligand binding to FPR/FPRL1 or CXCR, respectively.

Adult↗

PubMed2004

The phenylmethylthiazolylthiourea nonnucleoside reverse transcriptase (RT) inhibitor MSK-076 selects for a resistance mutation in the active site of human immunodeficiency virus type 2 RT.

The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.

Amino Acid Sequence↗

PubMed2003

The Drosophila melanogaster UMP-CMP kinase cDNA encodes an N-terminal mitochondrial import signal.

Drosophila melanogaster cells express a multi-substrate deoxyribonucleoside kinase that phosphorylates both purine and pyrimidine deoxyribonucleosides. The subsequent phosphorylation step is catalyzed by nucleoside monophosphate kinases. We have cloned and characterized the D. melanogaster UMP-CMP kinase (Dm.UMP-CMP kinase) to further study the nucleotide metabolizing enzymes in these cells. The kinase, encoded by the dak1 gene, was approximately 60% similar to the human UMP-CMP kinase and predominantly phosphorylated CMP, dCMP, and UMP. Expression analysis showed that the Dm.UMP-CMP kinase mRNA was constitutively expressed throughout the Drosophila development. The open-reading frame of the Dm.UMP-CMP kinase cDNA was extended in the 5'-region compared to UMP-CMP kinases of other species. The extended region encoded an N-terminal sequence with properties characteristic of a mitochondrial import signal. Expression of the enzyme in fusion with the green fluorescent protein verified that the N-terminal signal targeted the enzyme to mitochondria. This is the first time a mitochondrial pyrimidine nucleoside monophosphate kinase has been cloned from any organism and we discuss the implication of this finding for deoxyribonucleoside salvage in both Drosophila and other organisms.

Amino Acid Sequence↗

PubMed2003

Lipid-mediated protein delivery of suicide nucleoside kinases.

Nucleoside kinases from several species are investigated as suicide genes for treatment of malignant tumors by combined gene/chemotherapy. In the present study, we have investigated a novel strategy where nucleoside kinase proteins are directly delivered to cells without delivery of genetic material. We used a mix of a trifluoroacetylated lipopolyamine and dioleoyl phosphatidylethanolamine (BioPorter) to form protein-lipid complexes containing either recombinant herpes simplex virus type-1 thymidine kinase or Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. We showed that the nucleoside kinase containing protein-lipid complexes was imported into human osteosarcoma and Chinese hamster ovary cell lines by endocytosis and that the enzymes were delivered to the cytosol and nucleus. The nucleoside kinases imported into the cell lines retained enzymatic activity, and the cells treated with the enzyme-lipid complexes showed increased sensitivity to nucleoside analogues, such as ganciclovir, (E)-5-(2-bromovinyl)-2'-deoxyuridine, and 1-beta-D-arabinofuranosylthymine. Our results show that direct delivery of suicide gene proteins to cells may be an alternative approach to conventional suicide gene therapy strategies.

Animals↗

PubMed2003

Improving the selectivity of acyclic nucleoside analogues as inhibitors of human mitochondrial thymidine kinase: replacement of a triphenylmethoxy moiety with substituted amines and carboxamides.

Two series of analogues of the novel human mitochondrial thymidine kinase inhibitor 1-[(Z)-4-(triphenylmethoxy)-2-butenyl]thymine were synthesized by replacing the triphenylmethoxy moiety by a variety of substituted amines and carboxamides. In all the cases, the selectivity against the mitochondrial enzyme was either maintained or improved, and several derivatives were almost as potent as the parent compound. A molecular model was built that can account for the observed selectivities.

Enzyme Inhibitors↗

PubMed2003

Effects of 9-beta-D-arabinofuranosylguanine on mitochondria in CEM T-lymphoblast leukemia cells.

The nucleoside analog 9-beta-D-arabinofuranosylguanine (araG) is presently evaluated in clinical trials for therapy of T-cell lymphoid malignancies. AraG is a substrate for the mitochondrial deoxyguanosine kinase and we have recently shown that araG is predominantly incorporated into mitochondrial DNA (mtDNA). In this study we have investigated the effects of araG on mtDNA content and function. Although araG was incorporated into mtDNA, no decrease in mtDNA levels or effect on the expression of the mtDNA encoded cytochrome c oxidase was detected. Cells depleted of mtDNA were resistant to araG, but the mechanism of resistance was not specific for nucleoside analogs incorporated into mtDNA. Furthermore, the results suggest that the cells need to pass the S-phase in order for araG to induce caspase-dependent apoptosis. In summary, our findings suggest that the incorporation of araG into mtDNA does not cause the acute cytotoxicity of araG.

Antineoplastic Agents↗

PubMed2003

Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for therapy.

Nucleoside analogs act as prodrugs that must be converted to 5'-phosphates by intracellular kinases to become active in the treatment of viral and oncological diseases. Activation may be reversed by dephosphorylation if the 5'-phosphates are substrates for 5'-nucleotidases. Dephosphorylation by cytosolic enzymes decreases the efficacy of the analogs, whereas dephosphorylation by mitochondrial enzymes may decrease mitochondrial toxicity. Both effects may influence the outcome of therapy. We investigated the dephosphorylation of the 5'-phosphates of commonly used nucleoside analogs by two cytosolic (cN-II and dNT-1) and one mitochondrial (dNT-2) nucleotidase. Most uracil/thymine nucleotide analogs were dephosphorylated by all three human enzymes but cytosine-containing nucleotide analogs were inactive. Only cN-II showed some activity with the monophosphates of the two purine analogs 2-chloro-2'-deoxyadenosine and 9-beta-D-arabinosylguanine. We conclude that overproduction of any of the three 5'-nucleotidases cannot explain development of resistance against cytosine analogs but that overproduction of cN-II could lead to resistance against purine analogs. Of the tested analogs, only (E)-5-(2-bromovinyl)-2'-deoxyuridine was preferentially dephosphorylated by mitochondrial dNT-2. We propose that in future developments of analogs this aspect be considered in order to reduce mitochondrial toxicity. We tested inhibition of dNT-1 and dNT-2 by a large variety of synthetic metabolically stable nucleoside phosphonate analogs and found one (PMcP-U) that inhibited dNT-1 and dNT-2 competitively and a second (DPB-T) that inhibited dNT-2 by mixed inhibition. Both inhibitors are useful for specific 5'-nucleotidase assays and structural studies and may open up possibilities for therapy.

5'-Nucleotidase↗

PubMed2003

Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium.

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (<or=15 degrees C) leads to receptor deactivation/desensitization without any triggering of the superoxide anion-generating NADPH-oxidase. We show that the deactivated formyl peptide receptors (FPRs) can be reactivated/resensitized by the cytoskeleton-disrupting drug cytochalasin B. Such cytoskeleton-dependent receptor reactivation occurs also with the closely related receptors FPR-like-1 and C5aR but not with the receptors for interleukin-8 and platelet-activating factor. The reactivation state was further characterized with FPR as a model. The signals generated by receptor reactivation induced superoxide production that was terminated in 5-8 min, after which the neutrophils entered a new state of homologous deactivation. FPR antagonists were potent inhibitors of the superoxide production induced by the reactivated receptors, suggesting that the occupied receptors turn into an actively signaling state when the cytoskeleton is disrupted. The signals generated by the reactivated receptor were pertussis toxin-sensitive, indicating involvement of a G-protein. However, no transient elevation of intracellular Ca2+ accompanies the NADPH-oxidase activation. This was not due to a general down-regulation of phospholipase C/Ca2+ signaling, and despite the fact that no intracellular Ca2+ transient was generated, protein kinase C still appeared to be involved in the response. Further, phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and MEK all participated in the generation of second messengers from the reactivated receptors.

Androstadienes↗

PubMed2003

NADPH-oxidase activation in murine neutrophils via formyl peptide receptors.

Neutrophils play a key role at inflammatory sites where, in addition to destroying infecting microorganisms, they may also have deleterious effects on host tissues. Both activities involve activation of the NADPH-oxidase that produces bactericidal and tissue-destructive reactive oxygen species (ROS). We activated the murine NADPH-oxidase using different types of neutrophil activators and characterized the oxidative responses with respect to magnitude, localization, and kinetics. We show that agonist-induced activation of murine neutrophils results exclusively in extracellular release of ROS and no intracellular production could be detected. We also show that the formylated peptide, formyl-Met-Leu-Phe (fMLF), is a much less potent activator of the murine NADPH-oxidase than of the human analogue. Nevertheless, fMLF responses can be primed by pretreating the murine neutrophils with either cytochalasin B or bacterial lipopolysaccharide. Finally, we show that a synthetic hexapeptide, WKYMVM, is a more potent stimulus than fMLF for murine neutrophils and that these two agonists probably act via nonidentical high-affinity receptors.

Animals↗