Gene structure and regulation of the expression of the R1 and R2 subunits of mouse ribonucleotide reductase.
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Animals↗
Biomedical subjects
E Johansson
Publications and source records attributed to E Johansson.
Scientific rigour in qualitative research--examples from a study of women's health in family practice.
The increase in qualitative research in family medicine raises a demand for critical discussions about design, methods and conclusions. This article shows how scientific claims for truthful findings and neutrality can be assessed. Established concepts such as validity, reliability, objectivity and generalization cannot be used in qualitative research. Alternative criteria for scientific rigour, initially introduced by Lincoln and Guba, are presented: credibility, dependability, confirmability and transferability. These criteria have been applied to a research project, a qualitative study with in-depth interviews with female patients suffering from chronic pain in the locomotor system. The interview data were analysed on the basis of grounded theory. The proposed indicators for scientific rigour were shown to be useful when applied to the research project. Several examples are given. Difficulties in the use of the alternative criteria are also discussed.
Curriculum↗
Allergenic characterization of Acarus siro and Tyrophagus putrescentiae and their crossreactivity with Lepidoglyphus destructor and Dermatophagoides pteronyssinus.
Allergenic components in extracts of Acarus siro and Tyrophagus putrescentiae were identified by sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) and immunoblotting. Five and four allergenic components, respectively, were visualized with sera from farmers sensitized to storage mites. The highest frequency of IgE-binding was to a 15 kDa component of A. siro (7/9 sera) and a 16 kDa component of T. putrescentiae (23/29 sera). The allergenic crossreactivity of A. siro and T. putrescentiae with Lepidoglyphus destructor and Dermatophagoides pteronyssinus was studied with an immunoblotting inhibition technique. Crossreactivity between D. pteronyssinus on the one hand and A. siro and T. putrescentiae on the other, was shown, as the IgE reactivity to a 25 kDa component of D. pteronyssinus was inhibited to the same degree by extracts of A. siro, T. putrescentiae and D. pteronyssinus. However, D. pteronyssinus was a poor inhibitor of the allergenic components of A. siro and T. putrescentiae. Strong cross-reactivity was also shown between L. destructor and the allergenic components of A. siro and T. putrescentiae, while the latter mite species only to a very low degree inhibited the allergenic components of L. destructor.
Allergens↗
Oxygen-dependent modulation of release and activity of polymorphonuclear leukocyte granule products.
Polymorphonuclear leukocytes are important in the defense against the anaerobic microflora of infected gingival pockets. One part of this defense is release of antibacterial granule products by polymorphonuclear leukocytes into the pockets. The aim of the present study was to compare the efficiency of polymorphonuclear leukocytes in releasing granule products under aerobic and anaerobic conditions. Polymorphonuclear leukocytes were exposed to serum-opsonized zymosan under aerobic and anaerobic conditions. The levels of released granule products were determined by combining measurements of activity with enzyme-linked immunosorbent assays. The level of released elastase was twice as high in anaerobic as in aerobic reaction mixtures. A similar difference was not detected for myeloperoxidase. However, myeloperoxidase was inactivated after its release under aerobic conditions. The release of lactoferrin was an efficient under aerobic as under anaerobic conditions. The effect of aerobic conditions on the release of elastase and the inactivation of myeloperoxidase could be ascribed to oxidants formed in the myeloperoxidase-H2O2-chloride system. Also, the activity of the released cytoplasmic enzyme lactate dehydrogenase was inactivated by oxidants formed in the myeloperoxidase-H2O2-chloride system. These findings suggest that, in the anaerobic environment of the gingival pocket, elastase and possibly also other azurophilic granule products are released in higher amounts than under fully oxygenated conditions. In this environment, the released products may also escape inactivation by the myeloperoxidase-H2O2-chloride system.
Aerobiosis↗
Structure and promoter characterization of the gene encoding the large subunit (R1 protein) of mouse ribonucleotide reductase.
Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.
Animals↗
Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes.
Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes to kill a capsulated and a non-capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the capsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no detectable effect of 2 mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the beta-chain of C3b C3bi, and the C-terminal part of the alpha-chain of C3bi. This region of the alpha-chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The beta-chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system.
Anaerobiosis↗
Study of a reagent- and mediator-less biosensor for D-amino acids based on co-immobilized D-amino acid oxidase and peroxidase in carbon paste electrodes.
A biosensor for the analysis of D-amino acids is described. Carbon paste (graphite/paraffin oil) was chemically modified with immobilized D-amino acid oxidase and either horse-radish peroxidase or fungal peroxidase from Arthromyces ramosus. The two enzymes dissolved in buffer, together with an amine containing oligomer or polymer, were adsorbed on dry graphite. Prior to immobilization, the graphite was heat treated at 700 degrees C for 15 s to promote an efficient electron transfer between graphite and the peroxidase. The mixture was dried before addition of the pasting liquid. The sensor is based on the fact that the hydrogen peroxide produced by the action of D-amino acid oxidase is electrocatalytically reduced through the action of the peroxidase. The amine containing compound acted as a stabilizer and activator of the enzymes in the paste. The enzyme electrode was investigated as a sensor for D-phenylalanine and hydrogen peroxide in a flow through electrochemical cell connected to a single line flow injection system. The influences on the response by different additives to the paste and pH are reported. Linear calibration curves were obtained between 0.1 and 1.4 mM for D-phenylalanine and 5 and 1000 microM for hydrogen peroxide at an applied potential of -50 mV vs. Ag/AgCl. The sensor was also active for the following D-amino acids: D-alanine, D-valine, D-leucine, D-isoleucine, D-serine, D-aspartic acid, D-glutamic acid, D-lysine, D-histidine, D-arginine, D-tryptophan, D-methionine, and D-proline.
Amino Acids↗
Competitive solid-phase enzyme immunoassay for melatonin in human and rat serum and rat pineal gland.
We describe a solid-phase competitive enzyme immunoassay for determination of melatonin in serum. The detection limit of the assay is 1.0 fmol/well. Low cross-reactivity of the antiserum with other indoles, parallel serum extract dilution and melatonin standard curves, good analytical recovery, and within- and between-assay CVs of 6.4-14.4% provide validation of the assay. Linear regression analysis of melatonin concentrations measured with this assay (y) and with a commercial 3H RIA (x) in 88 sera yielded the relation y = 0.62 x - 0.76, Sy/x = 0.03. Values for melatonin in serum samples from healthy subjects are lower during the day than during the night. Melatonin response in rat serum and pineal gland to isoproterenol injection is similar to published RIA data. The analytical procedure is also simple. Thus, the assay should have practical applications in investigation of pineal function in both clinical and basic studies.
Animals↗
N-terminal aminoacid sequence of principal allergen of storage mite Lepidoglyphus destructor.
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Allergens↗
Age-associated changes and sex differences in urinary 6-sulphatoxymelatonin circadian rhythm in the rat.
The circadian rhythm of 6-sulphatoxymelatonin (aMT6s) excretion has been determined in male and female rats at 3 weeks and at 2, 8, 14 and 20 months of age. All animals have a pronounced circadian pattern of aMT6s excretion under a 12 hour dark: 12 hour light cycle. A significant increase in aMT6s excretion is observed from 3 weeks to 14 months followed by a decrease at 20 months. There is a highly significant correlation between aMT6s excretion and body weight (r = 0.73 for female rats and r = 0.74 for male rats; p values are all less than 0.001). Thus, a decrease in aMT6s excretion associated with increasing age occurs when body weight is taken into consideration. aMT6s excretion is higher in males at 3 weeks and at 2 and 8 months of ages. Urinary testosterone in male rats and estradiol in female rats increase from 3 weeks to 8 months and decrease at older ages. These data suggest that increase of body weight from 3 weeks to 14 months is an important factor responsible for the age-related alteration. The sex differences in aMT6s excretion in younger rats may be associated with their sex hormonal milieu.
Age Factors↗
Monoclonal antibodies to Lepidoglyphus destructor: delineation of crossreactivity between storage mites and house dust mites.
We have developed monoclonal antibodies (MoAbs) to the storage mite Lepidoglyphus destructor (Ld). Employing these anti-Ld MoAbs Ld-MoAbs) in ELISA and ELISA inhibition techniques we have analysed the reaction pattern of Ld-MoAbs to both non-pyroglyphid and pyroglyphid mites. The storage mite Glycyphagus domesticus (Gd) exhibited most efficient inhibition, followed by Acarus siro (As), Tyrophagus putrescentiae (Tp), Dermatophagoides pteronyssinus (Dpt) and Euroglyphus maynei (Em). Of the two pyroglyphid species, Dpt showed at least 1000 times less inhibition than Gd. Two of the MoAbs immunoprecipitated a band of 39 kD whereas the third reacted weakly, with a high-molecular band of approximately 110 kD. The Ld extract was also subjected to various reagents and conditions and the antigen was heat stable, it was not affected by low pH, or sensitive to dimethyl sulphoxide (DMSO) or paraformaldehyde. After exposure of the extract to various reagents, such as protease trypsin and periodate, we conclude that the epitopes recognized by Ld-MoAbs were of carbohydrate rather than of protein nature. It would thus seem that MoAbs recognize the carbohydrate part of a glycoprotein.
Animals↗
Screening of an elderly population in primary care for primary hyperparathyroidism.
In order to assess the diagnostic outcome of a screening for primary hyperparathyroidism (PHPT) in an elderly population, we determined ionized calcium in serum from 368 individuals participating in a health control at Mölnlycke Primary Care Centre (200 women, 168 men; age range 75-95 years); four-fifths of the individuals living in their homes, the remainder in homes for aged or nursing homes. Intact parathyroid hormone was determined in the samples with oinized calcium concentration greater than mean + 3SD of the truncated population sample, and these individuals were also recalled for another blood sample. Moderate hypercalcaemia, probably due to PHPT, was found in eight individuals (2% of the complete sample, 3% of the women), five having neuropsychiatric or neuromuscular symptoms consistent with PHPT. Surgical intervention is probably indicated in only a small proportion of elderly patients. We conclude that optimal benefits in relation to costs of screening for PHPT in old people will depend on the availability of a safe and simple pharmacological treatment that could determine any causal relationship between hypercalcaemia and symptoms.
Aged↗
Identification of a new major allergen of 39 kilodaltons of the storage mite Lepidoglyphus destructor.
The allergen composition of a non-denatured extract of the storage mite Lepidoglyphus destructor was studied by a combination of hybridoma technology, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a sandwich radio-allergosorbent test (four-step RAST). Using hybridoma technology, monoclonal antibodies (mAbs) were generated against the non denatured extract of the L. destructor. After screening by ELISA, mAb 42B6 was selected for further studies. This mAb specifically recognized an antigen of L. destructor of 39 kDa in SDS-PAGE. By a sandwich RAST, sera from 8 patients with known allergy to L. destructor were compared with control sera from 8 allergic patients. The results showed the presence of specific IgE against the 39-kDa protein in the sera of the test group, but not in the control group. There was also a good correlation observed between these data and the results obtained with the classical RAST. These results indicate that the newly identified antigen of 39 kDa is a major allergen of the storage mite Lepidoglyphus destructor.
Allergens↗
Immunoblot multi-allergen inhibition studies of allergenic cross-reactivity of the dust mites Lepidoglyphus destructor and Dermatophagoides pteronyssinus.
The allergenic similarity of the pyroglyphid mite D. pteronyssinus and the glycyphagid mite L. destructor was investigated with a new immunoblotting inhibition technique allowing simultaneous comparison of several allergens. Extracts of D. pteronyssinus and L. destructor were separated by SDS-PAGE and electroblotted to nitrocellulose (NC). A serum pool containing IgE specific to the major allergens in both mites was mixed with serially diluted extracts of D. pteronyssinus and L. destructor and incubated with the mite allergens of NC. The inhibition of the IgE binding to NC was evaluated by densitometric scanning and percentage inhibition was calculated. The IgE antibodies to the 25-kD component in D. pteronyssinus, were inhibited to the same degree by extracts of D. pteronyssinus and L. destructor. Another major allergen component in D. pteronyssinus (16 kD) was also inhibited by L. destructor extract but to a lesser degree: 400 times more of the heterologous than of the homologous extract was needed for 50% inhibition. To produce 50% of heterologous inhibition of the two major allergen components at 15 and 53 kD of L. destructor, 2000 and 10,000 times more respectively, of D. pteronyssinus than of L. destructor extract were needed. Two minor allergen components of L. destructor showed some cross-reactivity with D. pteronyssinus. However, L. destructor was a stronger inhibitor of D. pteronyssinus than vice versa, probably because the sera were obtained from persons more sensitized to L. destructor than to D. pteronyssinus.
Allergens↗
Storage mites dominate the fauna in Swedish barn dust.
The mite fauna in hay dust of 16 farms located on the island of Gotland was analysed to establish whether there is a clear relationship between environmental exposure to storage mites and development of allergic symptoms. Eight of these farms belonged to farmers with respiratory symptoms related to barn dust (RS) and eight to healthy farmers (H). All farmers had previously participated in an epidemiological survey of hypersensitivity to respiratory allergens. The symptomatic farmers had not only a positive RAST to storage mites but they developed more than a 20% fall in FEV1 when challenged with Lepidoglyphus destructor (L. destructor) extract. The asymptomatic farmers were all negative in RAST to storage mites. Mites were found on all farms and the degree of infestation varied enormously, mite counts ranging from 75-150,000 mites/g dust. Five genera were detected but the different mite species were not represented on all farms. L. destructor was the dominating species on 13 of 16 farms. Samples from RS farms contained a significantly higher number of mites than H farms. The results of this study are in good agreement with our earlier studies showing that L. destructor is the most important allergen causing symptoms from both upper and lower airways among farmers.
Agricultural Workers' Diseases↗
Effect of sulfide ions on complement factor C3.
In infected sites such as the gingival pockets of patients with periodontal disease, sulfide levels up to 1 mmol/liter may be reached. There is little information, however, on how sulfide may interact with the host defense. In a previous study (R. Claesson, M. Granlund-Edstedt, S. Persson, and J. Carlsson, Infect. Immun. 57:2776-2781, 1989), it was shown that polymorphonuclear leukocytes were able to kill bacteria in the presence of 1 mM sulfide. However, sulfide seemed to interfere with the opsonization of the bacteria. It has been claimed that sulfide may be toxic by splitting disulfide bonds of proteins. In the present study, serum was exposed to 2 mM sulfide under anaerobic conditions, and the capacity of sulfide to split disulfide bonds of 10 serum proteins involved in opsonization was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodetection of the proteins after blotting. Sulfide had a low capacity to split the disulfide bonds of most proteins. Sulfide had, however, a pronounced effect on the complement component C3 in the form of C3bi. Sulfide released the C-terminal region of the alpha chain from C3bi. When C3 opsonizes bacteria, it is this region of C3bi which binds to complement receptor 3 (CR3) of the polymorphonuclear leukocytes. If sulfide has the same effect on C3bi deposited on the bacterial surface as it has on C3bi in solution, it will annihilate the very important contribution of C3bi to opsonization.
Complement C3↗
A strategy for ranking environmentally occurring chemicals. Part VI. QSARs for the mutagenic effects of halogenated aliphatics.
A strategy for the systematic analysis and priority ranking of environmental chemicals has been applied to a class of 58 halogenated aliphatic hydrocarbons. A training set of ten compounds representing this class, was selected by statistical design. The training set compounds were then subjected to biological testing in the Salmonella typhimurium reverse mutation assay (Ames test). The measured biological data, recorded as dose-response curves, were analyzed to determine the mutagenic potency (slope of the initial portion) and the mutagen dose (MD 50) required to increase the number of revertants above the background by 50%. For each compound, four mutagenic potency estimates and four MD 50 values were determined, all originating from the tester strains TA 100 and TA 1535 with and without metabolic activation. The obtained responses were analyzed with multivariate techniques to give QSAR models relating the mutagenic potency data to the physico-chemical properties of the compounds. Finally, the derived QSARs were used to predict the mutagenic potencies and the MD 50S for the non-tested compounds in the class.
Chemical Phenomena↗