[Is it time to reintroduce remedial classes? Let us offer better chances to children with MBD/DAMP!].
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Attention Deficit Disorder with Hyperactivity↗
Biomedical subjects
L Lindqvist
Publications and source records attributed to L Lindqvist.
Purification and characterization of a new beta-lactamase from Bacteroides uniformis.
A beta-lactam-resistant Bacteroides uniformis strain was isolated from a clinical specimen. The strain produced large amounts of beta-lactamase and was resistant to penicillins and cephalosporins. The specific activity of the unpurified beta-lactamase was 4.8 U/mg of protein with nitrocefin as the substrate. The enzyme was purified 188-fold by Q-Sepharose, Sephacryl S-300, and Mono Q column passages. Kinetic parameters of the enzyme were determined by a micromethod performed in microtiter plates. beta-Lactamase was inhibited by cefoxitin and imipenem and hydrolyzed cephalosporins more rapidly than penicillins. The molecular weight was determined by sodium dodecyl sulfate-gradient gel electrophoresis to be 32,500, and the isoelectric point was 4.5.
Amino Acid Sequence↗
Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis.
Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.
Acute Disease↗
Transferases of O-antigen biosynthesis in Salmonella enterica: dideoxyhexosyltransferases of groups B and C2 and acetyltransferase of group C2.
The O antigen is a polymer of oligosaccharide units. O antigens differ in their sugar composition and glycosidic linkages, and genes responsible for O-antigen-specific biosynthesis are grouped in the rfb gene cluster. In this study, we identified two abequosyltransferase genes and an acetyltransferase gene in Salmonella enterica groups B and C2 by in vitro assay and identified paratosyl-, tyvelosyl-, and abequosyltransferase genes from S. enterica groups A and D and Yersinia pseudotuberculosis serovar IIA, respectively, by comparison.
Acetyltransferases↗
In vitro synthesis of CDP-d-abequose using Salmonella enzymes of cloned rfb genes. Production of CDP-6-deoxy-D-xylo-4-hexulose, CDP-3,6-dideoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-galactose, and isolation by HPLC.
In vitro enzymic synthesis of CDP-D-[U-14C]abequose, CDP-6-deoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-xylo-4-hexulose was achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP-6-deoxy-D-xylo-4-hexulose was isolated after 30 min reaction, using CDP-D-glucose, NAD and CDP-glucose 4.6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 90.6%). From that intermediate, CDP-3,6-dideoxy-D-xylo-4-hexulose was produced in a reaction using NADH and a crude extract containing the required enzymes. CDP-D-abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concentrations of the coenzymes. In a two-step reaction, CDP-D-glucose was first converted to CDP-6-deoxy-D-xylo-4-hexulose, then, following addition of the required coenzymes and enzymes, CDP-D-abequose was formed from this intermediary product in a 1-h incubation. Starting from 250 mg CDP-D-glucose, the molar yield of CDP-D-abequose after protein precipitation and HPLC was 82%, corresponding to more than 200 mg. CDP-D-[U-14C]abequose was synthesised from alpha-D-[U14C]glucose 1-phosphate and CTP using purified glucose-1-phosphate cytidylyltransferase in a reaction preceding the later steps. GC-MS and NMR revealed that the hexose part of the end product was 3.6-dideoxy-D-galactose (abequose) and that the corresponding intermediates were 4-keto-6-deoxy-D-xylo-hexose and 4-keto-3,6-dideoxy-D-xylo-hexose, respectively. The synthesized CDP-6-deoxy-D-xylo-4-hexulose exhibited the characteristic ultraviolet light absorption at 318 nm but no corresponding absorption was found for CDP-3,6-dideoxy-D-xylo-4-hexulose. A HPLC technique, where the four CDP-sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products.
Carbohydrate Epimerases↗
Purification, characterization, and high performance liquid chromatography assay of Salmonella glucose-1-phosphate cytidylyltransferase from the cloned rfbF gene.
We report the purification and characterization of glucose-1-phosphate cytidylyltransferase, the first of five enzymes committed to biosynthesis of CDP-D-abequose from Salmonella enterica strain LT2. The purification was greatly facilitated by using a cloned rfbF gene encoding this enzyme. Pure enzyme was obtained by 64-fold enrichment in three chromatography steps. The NH2-terminal sequence of the purified enzyme was in agreement with the sequence predicted from the nucleotide sequence of the rfbF gene. The SDS-polyacrylamide gel electrophoresis estimated subunit M(r) of 31,000 agrees well with the M(r) of 29,035 calculated from the amino acid composition deduced from the nucleotide sequence of the rfbF gene. The glucose-1-phosphate cytidylyltransferase catalyzes a reversible bimolecular group transfer reaction and steady-state kinetic measurements, including product inhibition patterns, indicate that this reaction proceeds by a "ping-pong" type of mechanism. The Km values for CTP, alpha-D-glucose 1-phosphate, CDP-D-glucose, and pyrophosphate are 0.28, 0.64, 0.11, and 1.89 mM, respectively.
Amino Acid Sequence↗
Quenching of merocyanine 540 triplet state by nitroxyl radicals in liposomal systems: a laser flash photolysis study.
Laser flash photolysis experiments were undertaken to investigate the interaction between stearic acid nitroxide spin probes and photoexcited merocyanine 540 (MC540) in dimyristoyl-L-alpha-phosphatidylcholine liposomes (membrane model). The measurements of the paramagnetic signal decay kinetics of four different spin-labelled stearic acids (n-DSA) show that the direct interaction between the dye and the probe is affected by the position of the nitroxyl group along the carbon chain. Laser flash photolysis results reveal a significant decrease in the MC540 triplet lifetime in the presence of n-DSA, the effect depending on the depth at which the nitroxyl moiety is localized in the bilayer. Previous results on the rate of disappearance of the electron spin resonance (ESR) nitroxide signal on continuous photolysis of the same systems do not show the same dependence on the localization of the nitroxyl moiety in the liposome. Although the MC540 triplet state seems to be implicated in the reaction process, the results suggest that ESR and laser flash experiments demonstrate two different kinds of mechanism.
Dimyristoylphosphatidylcholine↗
Intermediates in the oxidation of a dihydropyridine derivative of AZT related to AIDS dementia, studied by nanosecond laser photolysis.
Radicals of the AZT derivative 5'-(1,4-dihydro-1-methyl-3-pyridinylcarbonyl)-3'-azido-2',3'-dideo xythymidine) were obtained on exposure of this compound in alkaline aqueous solution to pulsed laser emission of lambda = 355 nm and pulsewidth 2 ns FWHM. The radical formation was shown to be due to photoelectron ejection from the pyridine group following two-step excitation of the compound. The radicals were found to deprotonate rapidly. Electrochemical analysis showed that the photoionization does not affect the azido group.
AIDS Dementia Complex↗
Glycosyl transferases of O-antigen biosynthesis in Salmonella enterica: identification and characterization of transferase genes of groups B, C2, and E1.
In Salmonella enterica, there is a great variety of O antigens, each consisting of a short oligosaccharide (the repeating unit) repeated many times. The O antigens differ in their sugar composition and glycosidic linkages. The genetic determinants of the O antigen are located in an rfb gene cluster, and some, including those of S. enterica O serogroups B, C2, and E1, have been cloned and sequenced. In this study of the glycosyltransferases which form the glycosidic linkages, we identify and characterize the four mannosyl and three rhamnosyl transferase genes of the three rfb gene clusters.
Carbohydrate Sequence↗
[Cryptococcal meningitis should be suspected in diffuse cerebral symptoms or indistinct meningitis].
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Adult↗
Enzymatic synthesis and isolation of thymidine diphosphate-6-deoxy-D-xylo-4-hexulose and thymidine diphosphate-L-rhamnose. Production using cloned gene products and separation by HPLC.
A two-step enzymatic synthesis of dTDP-L-rhamnose is developed using enzymes from sonicated extracts of cultures of Escherichia coli K12 strains harboring plasmids containing different parts of the rfb gene cluster of Salmonella enterica LT2. The intermediate dTDP-6-deoxy-D-xylo-4-hexulose was isolated after a 1-h reaction, using only dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 89%). In a two-step reaction using dTDP-D-glucose and dTDP-D-glucose 4,6-dehydratase in the first step, and with NADPH, dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase in the second hour of incubation, the dTDP-D-glucose was fully converted to dTDP-L-rhamnose. The hexoses of both products were identified by mass spectroscopy. The molar yield of dTDP-L-rhamnose, after protein precipitation, anion-exchange chromatography and desalting by gel chromatography, was 62%, corresponding to more than 150 mg, starting from 250 mg of dTDP-D-glucose. When stored lyophilysed under nitrogen, these products were found to be stable for several months. Both dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose have light absorption maxima at 267 nm, with molar absorption coefficients close to that of dTMP. However, the absorption coefficient of dTDP-6-deoxy-D-xylo-4-hexulose at the absorption maximum of 320 nm (specific for sugars containing keto groups) was found to be approximately 20% higher than values presented earlier. Furthermore, an HPLC technique is presented for determining the net activity of dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase and NADPH:dTDP-6-deoxy-L-lyxo-4-hexulose-4-reductase, based on separation of dTDP-6-deoxy-D-xylo-4-hexulose and dTDP-L-rhamnose. The HPLC technique is also suitable for determination of all the nucleotide components involved in the synthesis.
Bacterial Proteins↗
Effect of beta-lactamase inhibitors on beta-lactamases from anaerobic bacteria.
Three beta-lactamase inhibitors in clinical use--clavulanic acid, sulbactam and tazobactam--were investigated for their activity on beta-lactamases from Bacteroides uniformis, Clostridium butyricum and Fusobacterium nucleatum. Purification of the beta-lactamases was carried out by anion-exchange chromatography, gel filtration and FPLC. The inactivation of beta-lactamase activity was determined spectrophotometrically with nitrocefin as substrate. Various concentrations of the inhibitors were preincubated at 30 degrees C together with the enzyme for different periods of time before determination of the beta-lactamase activity. The three beta-lactamases tested were more susceptible to tazobactam than to clavulanic acid and sulbactam. Clavulanic acid and sulbactam reduced the enzyme activity of the Bacteroides uniformis beta-lactamase more effectively than the Clostridium butyricum and Fusobacterium nucleatum beta-lactamases.
Bacteria, Anaerobic↗
Purification and characterization of an imipenem hydrolysing metallo-beta-lactamase from Bacteroides fragilis.
An imipenem resistant beta-lactamase producing strain of Bacteroides fragilis was isolated from a clinical specimen. The specific activity of the unpurified beta-lactamase was 5.5 U/mg protein. The beta-lactamase was purified 60-fold by Q Sepharose, Sephacryl S-300 and Mono Q column passages. The strain was able to inactivate imipenem and cefoxitin in broth cultures. The enzyme hydrolysed imipenem more rapidly than ampicillin, benzylpenicillin, cephalothin and cefoxitin. The activity of the enzyme was Zn2+ dependent and was completely inhibited by EDTA. The inhibition was reversed by ZnSO4. Preincubation with the common beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam did not reduce the enzyme activity. The molecular weight was determined by sodium dodecyl sulfate gradient gel electrophoresis to be 31,000 Daltons and the isoelectric point was 4.5.
Bacteroides fragilis↗
Effect of clavulanic acid, sulbactam and tazobactam on three different beta-lactamases from Bacteroides uniformis, Clostridium butyricum and Fusobacterium nucleatum.
The effect of three beta-lactamase inhibitors, clavulanic acid, sulbactam and tazobactam used in clinical practice were compared for their activity against purified beta-lactamases from Bacteroides uniformis, Clostridium butyricum and Fusobacterium nucleatum. The enzymes from B. uniformis and C. butyricum were produced in fermenters under controlled growth conditions and the enzyme from F. nucleatum was produced in batch cultures. Purification of the beta-lactamases was achieved by anion-exchange chromatography, gel filtration and FPLC-technique. The degree of inactivation of beta-lactamase activity was determined spectrophotometrically with nitrocefin as the substrate. The inhibitors in various concentrations were preincubated at 30 degrees C together with the enzyme for different time periods (0.5-120 min) before determination of the remaining beta-lactamase activity. The inhibitors all decreased the activity of the beta-lactamases investigated. Clavulanic acid and sulbactam were capable of reducing the enzyme activity of the B. uniformis beta-lactamase more effectively than the C. butyricum and F. nucleatum beta-lactamases. All beta-lactamases tested were more susceptible to tazobactam than to clavulanic acid and sulbactam.
Bacteroides↗
Cadmium and zinc in Mytilus edulis L. from the Bothnian Sea and the northern Baltic proper.
Contents of Cd and Zn in the blue mussel, Mytilus edulis, collected on the coast of the southern Bothnian Sea and the northern Baltic proper, were determined using flame AAS. The observed concentrations ranged from 4.7 to 10.8 mg kg(-1) dw for Cd and from 121 to 215 mg kg(-1) dw for Zn. The Zn/Cd ratio was around 20 at a majority of the investigated locations. Cd exhibited an approximate two-fold gradual increase from south to north in the study area. It is likely that this increase was due largely to enhanced bioavailability for Cd at lower salinity. However, it is not possible to eliminate the influence from a generally elevated overall concentration of cadmium in the southern Bothnian Sea caused by natural and anthropogenic input. Compared to mussels from marine waters on the west coast of Sweden, Cd concentrations were 5-10 times higher, similarity indicating an influence of low salinity. Zn did not show these salinity correlated differences. The high Zn content found south of the outlet of the river Dalälven and gradually decreasing southwards was possibly caused by the high riverine input.
Journal Article↗
Progressive multifocal leukoencephalopathy in AIDS.
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Acquired Immunodeficiency Syndrome↗
Structure dynamics of the hemoglobin mutants Hb Hôtel Dieu, HbG Philadelphia, HbJ Mexico, Hb St. Mandé and Hb San Diego, studied by nanosecond-laser-flash photolysis.
The kinetics of the change from the carboxy to the deoxy conformation of the mutated hemoglobins mentioned in the title and of normal human adult hemoglobin were determined from measurements of light absorption changes occurring up to 50 microseconds after nanosecond-laser photodissociation of the corresponding CO complexes. The spectral evolution of the mutated hemoglobins was found to be similar in its main features to that of normal hemoglobin. The kinetics could be decomposed into two phases with rates 1.1-1.8 x 10(6) s-1 and 0.17-0.34 x 10(6) s-1 (except Hb St. Mandé which displayed only the faster phase). Study of the mutated subunits of HbJ Mexico (alpha subunit) and Hb Hôtel Dieu (beta subunit) showed that they convert exponentially to the stable deoxy state after photodeligation at the same rates as the corresponding subunits of normal Hb: 1.1 x 10(6) s-1 (alpha) and 0.3 x 10(6) s-1 (beta). The results indicate that there is no direct correlation between the kinetics of spectral relaxation in the time range studied and the oxygenation properties for these hemoglobins. However, there is some indication that the kinetics are dependent upon the region of mutation.
Hemoglobin J↗
Functional significance of aminoacyl-tRNA synthetase complex in the aminoacylation of tRNA(Leu) isoacceptors.
Aminoacyl-tRNA synthetases, partially purified from rat liver by two different methods, were used in vitro to study aminoacylation profiles of tRNA(Leu) isoacceptors. On the basis of molecular weights, one preparation was similar to the multienzyme complex of aminoacyl-tRNA synthetases, whereas the other apparently represents a partially disrupted complex. In the aminoacylation assay, the multienzyme complex produced a profile of leucyl-tRNA isoacceptors that was similar to those found in vivo and in liver perfusion experiments. The aminoacylation profile that was obtained with the partially disrupted complex varied with the enzyme and leucine concentration used. Especially one of the tRNA(Leu) species was poorly aminoacylated at low leucine and enzyme concentration. These experiments point out that attention should be paid to the nature of the aminoacyl-tRNA synthetase preparation in experiments in which isoacceptor profiles are studied in vitro.
Amino Acyl-tRNA Synthetases↗