Biomedical subjects

Anna Karlsson

Publications and source records attributed to Anna Karlsson.

53 records · Page 3Linked to original sources

PubMed2003

Substrate specificity and phosphorylation of antiviral and anticancer nucleoside analogues by human deoxyribonucleoside kinases and ribonucleoside kinases.

Structural analogues of nucleosides, nucleoside analogues (NA), are used in the treatment of cancer and viral infections. Antiviral NAs inhibit replication of the viral genome, whereas anticancer NAs inhibit cellular DNA replication and repair. NAs are inactive prodrugs that are dependent on intracellular phosphorylation to their pharmacologically active triphosphate form. The deoxyribonucleoside kinases (dNK) and ribonucleoside kinases (rNK) catalyze the first phosphorylation step, converting deoxyribonucleosides and ribonucleosides to their corresponding monophosphate form. The dNKs have been studied intensively, whereas the rNKs have not been as thoroughly investigated. This overview is focused on the substrate specificity, tissue distribution, and subcellular location of the mammalian dNKs and rNKs and their role in the activation of NAs.

Amino Acid Sequence↗

PubMed2003

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.

Amino Acid Sequence↗

PubMed2003

MAGIC-type polymer gel for three-dimensional dosimetry: intensity-modulated radiation therapy verification.

A new type of polymer gel dosimeter, which responds well to absorbed dose even when manufactured in the presence of normal levels of oxygen, was recently described by Fong et al. [Phys. Med. Biol. 46, 3105-3113 (2001)] and referred to by the acronym MAGIC. The aim of this study was to investigate the feasibility of using this new type of gel for intensity-modulated radiation therapy (IMRT) verification. Gel manufacturing was carried out in room atmosphere under normal levels of oxygen. IMRT inverse treatment planning was performed using the Helios software. The gel was irradiated using a linear accelerator equipped with a dynamic multileaf collimator, and intensity modulation was achieved using sliding window technique. The response to absorbed dose was evaluated using magnetic resonance imaging. Measured and calculated dose distributions were compared with regard to in-plane isodoses and dose volume histograms. In addition, the spatial and dosimetric accuracy was evaluated using the gamma formalism. Good agreement between calculated and measured data was obtained. In the isocenter plane, the 70% and 90% isodoses acquired using the different methods are mostly within 2 mm, with up to 3 mm disagreement at isolated points. For the planning target volume (PTV), the calculated mean relative dose was 96.8 +/- 2.5% (1 SD) and the measured relative mean dose was 98.6 +/- 2.2%. Corresponding data for an organ at risk was 34.4 +/- 0.9% and 32.7 +/- 0.7%, respectively. The gamma criterion (3 mm spatial/3% dose deviation) was fulfilled for 94% of the pixels in the target region. Discrepancies were found in hot spots the upper and lower parts of the PTV, where the measured dose was up to 11% higher than calculated. This was attributed to sub optimal scatter kernels used in the treatment planning system dose calculations. Our results indicate great potential for IMRT verification using MAGIC-type polymer gel.

Ascorbic Acid↗

PubMed2003

Non-nucleoside inhibitors of mitochondrial thymidine kinase (TK-2) differentially inhibit the closely related herpes simplex virus type 1 TK and Drosophila melanogaster multifunctional deoxynucleoside kinase.

5'-O-Trityl derivatives of thymidine (dThd), (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), and their acyclic analogs 1-[(Z)-4-triphenylmethoxy-2-butenyl]thymine (KIN-12) and (E)-5-(2-bromovinyl)-1-[(Z)-4-triphenylmethoxy-2-butenyl]uracil (KIN-52) have been synthesized and evaluated for their inhibitory activity against the amino acid sequence related mitochondrial dThd kinase (TK-2), herpes simplex virus type 1 (HSV-1) TK, and Drosophila melanogaster multifunctional 2'-deoxynucleoside kinase (Dm-dNK). Several compounds proved markedly inhibitory to these enzymes and represent a new generation of nucleoside kinase inhibitors. KIN-52 was the most potent and selective inhibitor of TK-2 (IC(50), 1.3 microM; K(i), 0.50 microM; K(i)/K(m), 0.37) but was not inhibitory against HSV-1 TK and Dm-dNK at 100 microM. As found for the alternative substrate BVDU, the tritylated compounds competitively inhibited the three enzymes with respect to dThd. However, whereas BVDU behaved as a noncompetitive inhibitor (alternative substrate) of TK-2 and HSV-1 TK with respect to ATP as the varying substrate, the novel tritylated enzyme inhibitors emerged as reversible purely uncompetitive inhibitors of these enzymes. Computer-assisted modeling studies are in agreement with these findings. The tritylated compounds do not act as alternative substrates and they showed a type of kinetics against the nucleoside kinases different from that of BVDU. KIN-12, and particularly KIN-52, are the very first non-nucleoside specific inhibitors of TK-2 reported and may be useful for studying the physiological role of the mitochondrial TK-2 enzyme.

Adenosine Triphosphate↗

PubMed2002

Acyclic nucleoside analogues as novel inhibitors of human mitochondrial thymidine kinase.

A series of acyclic nucleoside analogues of 5'-O-tritylthymidine have been synthesized and evaluated as potential human mitochondrial thymidine kinase (TK-2) inhibitors. In this series, the sugar moiety of the parent 5'-O-tritylthymidine has been replaced by aliphatic chains including (E)- and (Z)-butenol, butynol, or butanol. Among them the (Z)-butenyl derivative (10) showed an IC(50) against TK-2 of 1.5 microM, being 1 order of magnitude more potent than the parent 5'-O-tritylthymidine. This lead compound has been further modified by replacing the thymine base by other pyrimidine bases such as 5-iodouracil, 5-ethyluracil, 5-methylcytosine, 3-N-methylthymine, or 5,6-dihydrothymine, as well as by the purine base guanine. The trityl group has also been replaced by different aliphatic and aromatic acyl moieties including tert-butylacetyl, hexanoyl, decanoyl, and diphenylacetyl moieties. The evaluation of the compounds against TK-2 and the phylogenetically close HSV-1 TK has shown that the base moiety plays a crucial role in their interaction against these pyrimidine nucleoside kinases. Also, the presence of a lipophilic substituent, preferentially an aromatic moiety such as diphenylmethyl or triphenylmethyl, is required for efficient TK-2 inhibition. Whereas some compounds showed marked specificity for either TK-2 (i.e, the 5,6-dihydrothymine derivative, 26) or HSV-1 TK (i.e., the butynyl derivative, 11), some others, including the (Z)-and (E)-butenyl derivatives 10 and 12, showed significant inhibition against both enzymes. They also proved to be inhibitory against HSV-1 TK in intact human osteosarcoma cells that were transduced with the HSV-1 TK gene.

Catalysis↗

PubMed2002

Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils.

Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.

Animals↗

PubMed2002

Activation of the neutrophil nicotinamide adenine dinucleotide phosphate oxidase by galectin-1.

Galectins are a group of lactose-binding proteins widely distributed in nature. Twelve mammalian galectins have so far been identified, but their functions are to a large extent unknown. In this work we study galectin-1 in its interaction with human neutrophils, with regard to both cell surface binding and activation of the superoxide-producing NADPH-oxidase. We show that galectin-1 is able to activate the neutrophil NADPH-oxidase, provided that the cells have been primed by extravasation from the blood into the tissue, an activation pattern that is similar to that of galectin-3. Using in vitro priming protocols, the galectin-1 responsiveness was found to correlate to granule mobilization and galectin-1 binding to the cells, suggesting the presence of granule-localized receptors that are up-regulated to the cell surface upon priming. By galectin-1 overlay of fractionated neutrophils we identified potential galectin-1 receptor candidates localized in the membranes of the secretory vesicle and gelatinase granules. The binding of galectin-1 and galectin-3 to neutrophil proteins was compared, as were the dose dependencies for activation by the two lectins. The results suggest that, although similarities are found between the two galectins, they appear to activate the NADPH-oxidase using different receptors. In conclusion, galectin-1 appears to have proinflammatory functions, mediated through activation of the neutrophil respiratory burst.

Adjuvants, Immunologic↗

PubMed2002

Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles.

Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of azurophilic granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in azurophilic granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of azurophilic granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized azurophilic granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability.

Adult↗

PubMed2002

Immunostimulatory DNA induces degranulation and NADPH-oxidase activation in human neutrophils while concomitantly inhibiting chemotaxis and phagocytosis.

We examined the effects of oligodeoxynucleotides (ODN) with different structures and sequences on human neutrophil function. In lymphocytes and monocytes, the CpG-mediated immunostimulation is dependent on motif content, flanking sequences and DNA backbone composition. In neutrophils, however, native phosphodiester ODN were without effect regardless of CpG content, while backbone-substituted phosphorothioate ODN (PS-ODN) modulated neutrophil function in a sequence-independent manner. The neutrophil respiratory burst and degranulation of the specific and gelatinase granules were markedly increased by PS-ODN, as was the shedding of L-selectin. In contrast, neutrophil chemotaxis and phagocytosis were inhibited by PS-ODN. In summary, PS-ODN have both stimulatory and inhibitory effects on neutrophil function. This impact of PS-ODN on neutrophil function is unique and distinct from that exerted on other immune cells, with respect to both the identity of the activating DNA molecules and the regulation of the effector functions. These findings may have implications for the development of DNA-based immunotherapy and vaccination.

Adjuvants, Immunologic↗

PubMed2002

Galectins as inflammatory mediators.

Over the last decade a vast amount of reports have shown that galectin-1 and galectin-3 are important mediators of inflammation. In this review we describe how the galectins may be involved in several parts of the inflammatory process, including the recruitment of neutrophils into an infected tissue and the recognition and killing of bacteria by activation of the tissue destructive phagocytic respiratory burst. During bacterial infection or aseptic inflammatory processes, galectins are produced and released by e.g. infected epithelium, activated tissue-resident macrophages and endothelial cells. These extracellular galectins may facilitate binding of neutrophils to the endothelium by cross-linking carbohydrates on the respective cells. Further the galectins improve binding of the neutrophil to the extracellular matrix proteins laminin and fibronectin, and are potential chemotactic factors, inducing migration through the extracellular matrix towards the inflammatory focus. When the cells encounter bacteria, galectin-3 could function as an opsonin, cross-linking bacterial lipopolysaccharide or other carbohydrate-containing surface structures to phagocyte surface glycoconjugates. Both galectin-1 and galectin-3 have the capacity to induce a respiratory burst in neutrophils, provided that the cells have been primed by degranulation and receptor upregulation. The reactive oxygen species produced may be destructive to the invading micro-organisms as well as to the surrounding host tissue, pointing out the possible role of galectins, not only in defence toward infection, but also in inflammatory-induced tissue destruction.

Animals↗

PubMed2002

Ionomycin-induced neutrophil NADPH oxidase activity is selectively inhibited by the serine protease inhibitor diisopropyl fluorophosphate.

The calcium-specific ionophore ionomycin triggers neutrophils to activate their NADPH oxidase and generate reactive oxygen species. This activation is restricted to intracellular sites and involves the neutrophil granules. Cells that have experienced an ionomycin-induced rise in intracellular calcium will also mobilize their intracellular granules and are primed to subsequent challenge with the chemoattractant formylmethionyl-leucyl-phenylalanine (fMLF), but have lost their ability to become desensitized to the same agonist. We have investigated the involvement of serine proteases in the calcium-induced effector functions using the inhibitor diisopropyl fluorophosphate (DFP). The ionomycin-induced NADPH oxidase activity was abrogated by the protease inhibitor, whereas the activity induced by fMLF was unaffected. The DFP-dependent inhibition was restricted to the NADPH oxidase activity, as all other ionomycin-induced cellular activities were largely unaffected. We thus suggest that a serine protease is of importance for the calcium ionophore-induced signal(s) to reach and activate the dormant NADPH oxidase in the neutrophil granules.

Cell-Free System↗

PubMed2002

Assembly and activation of the neutrophil NADPH oxidase in granule membranes.

Phagocyte activation is accompanied by assembly of an NADPH oxidase that reduces oxygen to form a number of reactive species. These oxygen radicals can eradicate invading microorganisms, regulate the function of other immune reactive cells, and cause damage to "innocent bystander" cells. It is generally assumed that the NADPH oxidase is activated exclusively in the plasma membrane. In neutrophils, this assumption does not fit with the subcellular localization of the membrane component of the oxidase, which is stored in granule compartments. It has now become increasingly evident that oxidants are also produced in an intracellular compartment that we identify as the specific granules. Myeloperoxidase is stored in another granule subset, the azurophil granules, and participates in the processing of the oxidative metabolites. We suggest that neutrophil activation is accompanied by fusion between azurophil and specific granules, allowing these peroxidase-dependent reactions to take place. The presented data suggest a requisite role for neutrophil oxidants complementing their function as microbial killing agents. Signaling capabilities of the oxidants, affecting for example, the state of protein phosphorylation, regulation of transcription factors, and induction of apoptosis, are discussed.

Animals↗

PubMed2002

Nucleotide affinity for a stable phosphorylated intermediate of nucleoside diphosphate kinase.

Nucleoside diphosphate (NDP) kinase is transiently phosphorylated on a histidine of the active site during the catalytic cycle. In the presence of a nucleotide acceptor, the phosphohistidine bond is unstable and the phosphate is transferred to the acceptor in less than 1 msec. We describe the synthesis of an analog of the phosphoenzyme intermediate with an inactive mutant of NDP kinase in which the catalytic histidine is replaced by a cysteine. In two sequential disulfide exchange reactions, a thiophosphate group reacts with the thiol function of the cysteine that had previously reacted with dithionitrobenzoate (DTNB). The thiophosphoenzyme presents a 400,000-fold increased stability in the presence of NDPs compared with the phosphoenzyme. The binding of NDP is studied at the steady state and presteady state. Data were analyzed according to a bimolecular association model. For the first time, the true equilibrium dissociation constants of NDP for the analog of the phosphoenzyme are determined in the absence of phosphotransfer, allowing a better understanding of the catalytic mechanism of the enzyme.

Amino Acid Substitution↗

PubMed2002

Lipopolysaccharide-induced granule mobilization and priming of the neutrophil response to Helicobacter pylori peptide Hp(2-20), which activates formyl peptide receptor-like 1.

The cecropin-like bactericidal peptide Hp(2-20) from Helicobacter pylori induces activation of the NADPH oxidase in human neutrophils via formyl peptide receptor-like 1 (FPRL1) (J. Bylund, T. Christophe, F. Boulay, T. Nyström, A. Karlsson, and C. Dahlgren, Antimicrob. Agents Chemother. 45:1700-1704, 2001). Here we investigated the ability of bacterial lipopolysaccharide (LPS) to prime this response. Neutrophils treated with LPS for 30 min at 37 degrees C produced substantially more superoxide anion than control cells upon stimulation with Hp(2-20). Hence, LPS primed the cells for subsequent stimulation through FPRL1. To study the molecular background of this priming phenomenon, we measured the degrees of granule mobilization and concomitant receptor upregulation to the cell surface in LPS-treated cells. Exposure of complement receptors 1 and 3 as well as the formyl peptide receptor (FPR) was markedly increased after LPS treatment. Since approximately 60% of the gelatinase granules were mobilized while the specific granules were retained, we hypothesized that the gelatinase granules were potential stores of FPRL1. The presence of FPRL1 mainly in the gelatinase granules was confirmed by Western blotting of subcellular fractions of resting neutrophils. These results suggest that the mechanism behind the LPS-induced priming of FPRL1-mediated responses lies at the level of granule (receptor) mobilization.

Amino Acid Sequence↗

PubMed2002

Variation in extracellular protease production among clinical isolates of Staphylococcus aureus due to different levels of expression of the protease repressor sarA.

Staphylococcus aureus produces four major extracellular proteases: staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp). Several in vitro studies have suggested that these enzymes are important virulence factors. Here we analyzed the protease production of 92 S. aureus strains from infected human soft tissue. Twenty-one strains produced variable zones of proteolysis on casein agar plates, while the remaining 71 strains appeared to be protease negative. The major protease genes were present in all protease-positive (n = 5) and protease-negative (n = 12) strains analyzed. Northern blotting showed that transcription of the protease genes was suppressed due to increased sigma factor B (SigB)-dependent expression of the protease repressor SarA. Other SigB-dependent traits such as pigmentation and expression of asp 23 were also increased in protease-negative compared to protease-positive strains. Inactivation of sarA in three protease-negative strains resulted in increased transcription of all protease genes and increased protease production, while overexpression of sarA in a strain producing protease at high levels repressed protease production. Our results suggest that the protease genes are conserved among clinical S. aureus strains and that the level of SigB-dependent expression of the protease repressor sarA determines the level of protease production in each strain.

Bacterial Proteins↗

PubMed2002

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase.

The neutrophil NADPH-oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane-localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF-induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin-induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose-dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 microM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels.

Calcium↗

PubMed2002

The presence of stomatin in detergent-insoluble domains of neutrophil granule membranes.

Neutrophil azurophil granules, traditionally regarded as the neutrophil counterpart to lysosomes, lack the lysosomal marker lysosome-associated membrane glycoprotein and have recently been suggested to be nonlysosomal secretory organelles. The membrane of the azurophil granules is poorly characterized-CD63 and CD68 are the only membrane proteins identified so far. Here, azurophil granule membranes were isolated by Percoll gradient subcellular fractionation. Using matrix-assisted laser desorption ionization time of flight mass spectrometry of tryptic peptides from an isolated protein, stomatin was identified in these membranes. Using immunoelectron microscopy and immunoblot analysis of isolated organelles, stomatin was found to be subcellularly localized, not only to the azurophil granules but also by a major part to the specific granules and by a minor part to the secretory vesicles/plasma membrane. We also show the presence of detergent-insoluble, low-density membrane domains in the plasma membrane and the granule membranes and found stomatin to be localized to these domains.

Blood Proteins↗