Chapter 3. Major health problems. Health in Sweden: The National Public Health Report 2001.
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Adolescent↗
Biomedical subjects
A Karlsson
Publications and source records attributed to A Karlsson.
Allergic disease and the infestation of Enterobius vermicularis in Swedish children 4-10 years of age.
BACKGROUND: The possible association between certain childhood infections and the propensity to develop allergic disease may include intestinal helminth infections. The experiences of such associations derive mainly from studies in tropical areas, and the results are not clear-cut. OBJECTIVE: To explore the association between Enterobius vermicularis and allergic disease in Swedish children 4 to 10 years of age. METHOD: The occurrence of E. vermicularis was examined by perianal tape tests in 70 allergic children recovered from a pediatric register of positive skin-prick tests. A nonallergic control group (n = 102) was gathered from a cohort of children previously examined for the prevalence of E. vermicularis and allergic symptoms. RESULTS: In the allergic group 26 of the 70 cases (37%) had a positive tape-test for E. vermicularis, compared to 23 of the 102 cases (23%) in the nonallergic control group (p = .037). CONCLUSION: The results indicate that E. vermicularis could be more frequent in children with allergic disease as defined by allergic symptoms and a positive skin-prick test compared to nonallergic children, i. e., those without a history of allergic disease. These data, however, do not allow any conclusion on the nature of the possible association between E. vermicularis and allergic disease.
Animals↗
Conserved gene structure and transcription factor sites in the human and mouse deoxycytidine kinase genes.
Deoxycytidine kinase (dCK) phosphorylates several anti-cancer and anti-viral nucleoside analogs. The enzyme is predominantly expressed in lymphoid tissues regulated by an unknown mechanism. We have cloned and sequenced the 20 kbp mouse dCK gene and approximately 1.7 kbp of the 5' flanking regions of both the human and mouse dCK genes. Five major inter-species conserved motifs were identified in the 5' region including the transcription initiation region, an SP1 site and two closely located putative octamer transcription factor sites. Luciferase reporter experiments showed that the human dCK 5' region efficiently initiated transcription but no tissue regulatory element could be identified.
5' Untranslated Regions↗
Retroviral transduction of cancer cell lines with the gene encoding Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.
Nucleoside kinases from several species are investigated as "suicide genes" for treatment of malignant tumors by combined gene/chemotherapy. We have recently cloned a multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK), and we have shown that the enzyme phosphorylates cytotoxic pyrimidine and purine nucleoside analogs. The broad substrate specificity of the enzyme, as well as its very high catalytic rate, makes it a unique member of the nucleoside kinase enzyme family. In the present study, we evaluated Dm-dNK as a suicide gene by constructing a replication-deficient retroviral vector that expresses the enzyme. The human pancreatic adenocarcinoma cell line MIA PaCa-2 and a thymidine kinase-deficient osteosarcoma cell line were transduced with the recombinant virus. We showed that Dm-dNK can be expressed in human cells, that the enzyme retained its enzymatic activity, and that it is localized in the cell nuclei due to a nuclear localization signal in its C-terminal region. The cells expressing Dm-dNK exhibited increased sensitivity to several cytotoxic nucleoside analogs, such as 1-beta-d-arabinofuranosylcytosine, 1-beta-d-arabinofuranosylthymine, (E)-5-(2-bromovinyl)-2'-deoxyuridine, 2-chloro-2'-deoxyadenosine, and 2',2'-difluorodeoxycytidine. These findings suggest that Dm-dNK may be used as a suicide gene in combined gene/chemotherapy of cancer.
Adenocarcinoma↗
Novel ribofuranosylnucleoside lead compounds for potent and selective inhibitors of mitochondrial thymidine kinase-2.
The ribonucleoside analogues (E)-5-(2-bromovinyl)uridine (5-BV-Urd) and 3'-spiro-(4'-amino-1',2'-oxathiole-2',2'-dioxide)-5-methyluridine (3'-AOD-5-MeUrd) emerged as potent and selective competitive inhibitors of mitochondrial thymidine kinase (TK)-2 with respect to thymidine (K(i)/K(m) values of 9.0 and 1.2 respectively). Cytosolic TK-1 did not show measurable affinity for these compounds. [(32)P]Phosphate transfer studies from [gamma-(32)P]ATP to 5-BV-Urd and 3'-AOD-5-MeUrd revealed extremely poor substrate activity but potent inhibitory potential of the compounds. It was concluded that the ribonucleosides 5-BV-Urd and 3'-AOD-5-MeUrd represent two new lead compounds for potent and selective inhibitors of mitochondrial TK-2.
Adenosine Triphosphate↗
The subcellular location of nucleoside analog phosphorylation is a determinant of synergistic effects of hydroxyurea.
The ribonucleotide reductase inhibitor hydroxyurea exhibits synergistic pharmacological activity with several nucleoside analogs used in antiviral and anticancer chemotherapy. We have used a cell model system where a deoxycytidine kinase (dCK)-deficient cell line was reconstituted with genetically engineered dCK targeted to the cytosol, the nucleus, or the mitochondria to investigate how the subcellular location of nucleoside analog phosphorylation affected the synergistic effects of a ribonucleotide reductase inhibitor. Hydroxyurea showed synergistic cytotoxicity with the nucleoside analogs 1-beta-d-arabinofuranosylcytosine and 2-chloro-2'-deoxyadenosine when dCK was expressed in the cytosol or in the nucleus, but not when dCK was expressed in the mitochondria. These data indicate that the synergistic effect of ribonucleotide reductase inhibition is limited to nucleoside analogs phosphorylated in the cytosol or the cell nucleus.
Animals↗
Incorporation of nucleoside analogs into nuclear or mitochondrial DNA is determined by the intracellular phosphorylation site.
Nucleoside analogs used in cancer chemotherapy and in treatment of virus infections are phosphorylated in cells by nucleoside and nucleotide kinases to their pharmacologically active form. The phosphorylated nucleoside analogs are incorporated into DNA and cause cell death or inhibit viral replication. Cellular DNA is replicated both in the nucleus and in the mitochondria, and nucleoside analogs may interfere with DNA replication in both these subcellular locations. In the present study we created a cell model system where nucleoside analogs were phosphorylated, and thereby pharmacologically activated, in either the nucleus, cytosol, or mitochondria of cancer cells. The system was based on the reconstitution of deoxycytidine kinase (dCK)-deficient Chinese hamster ovary cells with genetically engineered dCK targeted to the different subcellular compartments. The nucleoside analogs phosphorylated by dCK in the mitochondria were predominantly incorporated into mitochondrial DNA, whereas the nucleoside analogs phosphorylated in the nucleus or cytosol were incorporated into nuclear DNA. We further show that the nucleoside analogs phosphorylated in the mitochondria induced cell death by an apoptotic program. These data showed that the subcellular site of nucleoside analog phosphorylation is an important determinant for incorporation of nucleoside analogs into nuclear or mitochondrial DNA.
Amino Acid Sequence↗
Packed-column supercritical fluid chromatography for the analysis of isosorbide-5-mononitrate and related compounds in bulk substance and tablets.
We describe a packed-column supercritical fluid chromatographic method that can be used for the analysis of isosorbide-5-mononitrate (5-ISMN) bulk substance and the 5-ISMN content of Imdur tablets. The method is based on methanol-modified carbon dioxide as the mobile phase and porous graphitized carbon (PGC, Hypercarb) as column support at 40 degrees C and 100 bar back pressure. The method makes it possible to simultaneously determine 5-ISMN and related compounds. In order to elute NO(3)(-) with acceptable retention time a quarternary ammonium hydrogen sulfate salt is added to the methanol modifier. An almost linear increase of the retention time with increasing carbon content of the counter ion was found. Tetramethyl ammonium hydrogen sulfate 5 mM in methanol was used in the final method as polar modifier for the simultaneous determination of possible degradation products within 12 min. The present method can separate and detect related compounds such as isosorbide-2, 5-dinitrate, isomannidemononitrate and isosorbide-2-mononitrate at the 0.1% (w/w) level as required by regulatory guidelines. Nitrate can be detected down to about 0.02% (w/w). Repeated analyses of ground tablet powder gave an assay precision for isosorbide-5-mononitrate of 1.4% (R.S.D., eight samples and two injections of each). For related substances at an area percent of 0. 1 the precision was less than 10%.
Chromatography↗
Differential incorporation of 1-beta-D-arabinofuranosylcytosine and 9-beta-D-arabinofuranosylguanine into nuclear and mitochondrial DNA.
The anti-leukemic nucleoside analogs 1-beta-D-arabinofuranosylcytosine (araC) and 9-beta-D-arabinofuranosylguanine (araG) are dependent on intracellular phosphorylation for pharmacological activity. AraC is efficiently phosphorylated by deoxycytidine kinase (dCK). Although araG is phosphorylated by dCK in vitro, it is a preferred substrate of mitochondrial deoxyguanosine kinase. We have used autoradiography to show that araC was incorporated into nuclear DNA in Molt-4 and CEM T-lymphoblastoid cells as well as in Chinese hamster ovary cells. In contrast, araG was predominantly incorporated into mitochondrial DNA in the investigated cell lines, without detectable incorporation into nuclear DNA. These data suggest that the molecular targets of araG and araC may differ.
Animals↗
The human nm23-H4 gene product is a mitochondrial nucleoside diphosphate kinase.
We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.
Base Sequence↗
Ion-exclusion controlled size-exclusion chromatography of methacrylic acid-methyl methacrylate copolymers.
Controlled ion-exclusion allows compensation of hydrophobic adsorption in size-exclusion chromatography of negatively charged methacrylic acid-methyl methacrylate (Eudragit) polymers using methanol as a mobile phase. Properly selected low-ionic-strength conditions below 5 mM LiCl provide correct separation in the size-exclusion mode. Possible disturbing effects, mainly related to light scattering, under low-salt conditions are discussed and shown to be negligible if on-line concentration-light scattering detection is used. The absence of these disturbances is checked by a comparison of experiments performed in methanol containing 1.25 mM and 2.5 mM LiCl. Molecular mass averages and distributions identical within the experimental error are obtained.
Chromatography, Gel↗
The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A(4) receptor.
A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[(3)H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA(4)R because it has been shown to bind lipoxin A(4), we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC(50 )of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC(50 )of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA(4)R like FPR is stored in mobilizable organelles. (Blood. 2000;95:1810-1818)
Calcium↗
Proline-specific dipeptidyl peptidase activity in the cockroach brain and intestine: partial characterization, distribution, and inactivation of tachykinin-related peptides.
Proline-specific dipeptidyl peptidase (DPP IV) is an established enzyme known to degrade neuropeptides and peptide hormones in vertebrate tissues. DPP IV cleaves peptides at the Pro2 residue. Because several neuropeptides of the cockroach Leucophaea maderae, such as LemTRP-1 (APSGFLGVRamide), are potential substrates for this peptidase, we investigated the occurrence of proline-specific DPP activity in cockroach tissues. Partly purified DPP activity was characterized from the brain and midgut of L. maderae by using Gly-Pro-4-nitroanilide as a substrate. The highest activity was obtained from the membrane fraction of intestine; about 10 times less activity (per milligram protein) was obtained from brain membranes. A smaller amount of soluble DPP activity could also be identified in both tissues. Gel chromatography of the solubilized intestinal DPP activity revealed a molecular mass of about 75 kDa. The enzyme had a pH optimum of 8.5. Diprotin A (Ile-Pro-Ile) was an efficient competitive inhibitor of the cockroach DPP, whereas other known DPP inhibitors were found to be less potent. When incubated with human and cockroach DPP IV, the cleavage products of LemTRP-1 were AP and SGFLGVRamide (des-AP-LemTRP-1) as determined by mass spectrometry of high-performance liquid chromatography (HPLC)-purified peptide fragments. The AP fragment was biologically inactive and the des-AP fragment had a drastically reduced myostimulatory activity on the hindgut of L. maderae. The blowfly TRP callitachykinin-I (CavTK-I; APTAFYGVRamide) was cleaved in two steps to des-AP-CavTK-I and desAPTA-CavTK-I, showing that cockroach DPP does not only liberate Xaa-Pro, but also Xaa-Ala dipeptides. The fragment desAPTA-CavTK-I was completely inactive on the cockroach hindgut. To compare, LemTRP-3 and CavTK-II, which lack a Pro2, were not cleaved by DPP IV. Enzyme histochemistry for DPP IV was performed on cryostat sections of brain and intestine with Gly-Pro-4-methoxy-2-naphthylamide as the substrate and Fast Blue B as the chromogen. Strong histochemical labeling was seen in specific neuropils of the brain such as the calyces of the mushroom bodies, the antennal glomeruli, and the central body. Also, the inner lining of the midgut (the peritrophic membrane) and the malpighian tubules were strongly labeled by reaction product. In both the brain and intestine, the enzyme-histochemical reaction was inhibited by diprotin A.
Animals↗
Mammalian 5'(3')-deoxyribonucleotidase, cDNA cloning, and overexpression of the enzyme in Escherichia coli and mammalian cells.
5'(3')-Deoxyribonucleotidase is a ubiquitous enzyme in mammalian cells whose physiological function is not known. It was earlier purified to homogeneity from human placenta. We determined the amino acid sequences of several internal peptides and with their aid found an expressed sequence tag clone with the complete cDNA for a murine enzyme of 23.9 kDa. The DNA was cloned into appropriate plasmids and introduced into Escherichia coli and ecdyson-inducible 293 and V79 cells. The recombinant enzyme was purified to homogeneity from transformed E. coli and was found to be identical with the native enzyme. After induction with ponasterone, the transfected mammalian cells showed a gradual increase of enzyme activity. A human expressed sequence tag clone contained a large part of the cDNA of the human enzyme but lacked the 5'-end corresponding to 51 amino acids of the murine enzyme. Several polymerase chain reaction-based approaches to find this sequence met with no success. A mouse/human hybrid cDNA that had substituted the missing human 5'-end with the corresponding mouse sequence coded for a fully active enzyme.
5'-Nucleotidase↗
Substrate binding site of naphthalene 1,2-dioxygenase: functional implications of indole binding.
The three-dimensional structure of the aromatic hydroxylating enzyme naphthalene dioxygenase (NDO) from Pseudomonas sp. NCIB 9816-4 was recently determined. The refinement of the structure together with cyclic averaging showed that in the active site of the enzyme there is electron density for a flat aromatic compound. This compound appears to be an indole adduct, which in Escherichia coli is derived from tryptophan present in the rich culture medium. An indole-dioxygen adduct has been built which fits the electron density convincingly. Support for this interpretation was obtained from crystals of the enzyme purified from cells grown in the absence of tryptophan which had an empty substrate pocket. These types of crystals were soaked in indole solutions and the position of indole in this complex was similar to the corresponding part in the modelled indole-oxygen adduct. This suggests that a peroxide bound to iron end-on attacks the substrate and forms this intermediate. The substrate position has implications for the substrate specificity of the enzyme. Docking studies with indole, naphthalene and biphenyl inside the substrate pocket of NDO suggest the presence of subpockets where the one close to the active site iron is reserved for the binding of the aromatic ring which is hydroxylated upon catalysis. The plausible location for the binding of dioxygen is between this pocket and the catalytic iron. This is in accordance with the enantiospecificity of the products.
Amino Acid Sequence↗
The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.
Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.
Crystallization↗
Enantiomeric separation of amino alcohols on protein phases using statistical experimental design. A comparative study.
Two LC supports often used for giving enantioselective retention were tested and compared in the reversed-phase mode using statistical experimental design. The two supports contain two different proteins, alpha1-acid glycoprotein or cellulase immobilised to silica particles, as the chiral selectors. The two chromatographic columns are commercially available as Chiral-AGP and Chiral-CBH. Twelve closely structurally related amino alcohols were used as the testing solutes. For each column three important mobile phase descriptors, that improve the chiral recognition, were chosen as variables and retention and separation factors were used as responses. All the tested solutes were separated using the two protein based supports. However, the highest enantioselectivities, i.e., separation factors higher than 10 were obtained using the Chiral-CBH column. The solute structure, e.g., distance between the nitrogen atom and the chiral carbon atom, and position as well as type of substituent in the aromatic ring highly influence the enantioselectivity on both columns. For one of the solutes the choice of mobile phase composition could be used to control the retention order of the two enantiomers.
Amino Alcohols↗
Polar organic phase liquid chromatography with packed capillary columns using a vancomycin chiral stationary phase
Vancomycin immobilized on silica served as the chiral stationary phase (CSP) in this investigation with polar organic solvents as the mobile phase in liquid chromatography (LC). It was shown that trace amounts of water were beneficial for improving peak shape and efficiency. To regulate the retention and selectivity an acid and/or base were added to the mobile phase where an excess of acid was shown to be preferential for enantioseparation. An unusual increase in selectivity with increasing temperature was shown for the acidic drug, thalidomide. Additionally, nonlinear van't Hoff plots were obtained for metoprolol enantiomers that showed increased retention with increasing temperature. Metoprolol also showed unusual behavior in the polar organic phase when water was added to resemble reversed-phase chromatography, with minimum retention observed at high water or high methanol concentrations. In both instances a high degree of electrostatic interaction between metoprolol and vancomycin was concluded. Metoprolol and ten of its analogs were examined on this CSP to evaluate the enantiorecognition process. A comparison in enantioselectivity for a number of acidic and basic drugs using this CSP was also carried out using the polar organic phase, reversed phase, and normal phase LC which were all compared to the results obtained in supercritical fluid chromatography (SFC). Polar organic phase LC offered a better separation of basic molecules while reversed phase LC was preferred for the resolution of acids. SFC showed the broadest enantioselectivity overall and normal phase LC indicated similar properties, as expected, to SFC but with lower column efficiency. Copyright 2000 Wiley-Liss, Inc.
Journal Article↗