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Biomedical subjects

K Andersson

Publications and source records attributed to K Andersson.

At least 19 recordsLinked to original sources
PubMed2000

Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels.

The interactions between a set of drugs, selected on the basis of reported human serum albumin (HSA) binding levels, and immobilized HSA were investigated using surface plasmon resonance technology. Major HSA binding sites were available after immobilization. The intensity of the signal obtained from the interaction of the drug with the HSA surface was correlated with the reported HSA binding level. Drugs were classified into groups corresponding to high, medium, or low HSA binding based on the injection of the drug at 80 microM concentration. A set of 10 drugs binding to alpha(1)-acid glycoprotein (AGP) was also investigated and correlated with reported AGP binding data. The throughput of the presented assay is 100 compounds/24 h, and the sample consumption is less than 100 microL (8 nmol).

Binding Sites↗
PubMed2000

A missense mutation in the heavy subunit of gamma-glutamylcysteine synthetase gene causes hemolytic anemia.

gamma-Glutamylcysteine synthetase (GCS) catalyzes the initial and rate-limiting step in the biosynthesis of glutathione. gamma-GCS consists of a heavy and a light subunit encoded by separate genes. Hereditary deficiency of GCS has been reported in 6 patients with hemolytic anemia and low erythrocyte levels of glutathione and gamma-glutamylcysteine. In addition, 2 patients also had generalized aminoaciduria and developed neurologic symptoms. We have examined a Dutch kindred with 1 suspected case of GCS deficiency. The proband was a 68-year-old woman with a history of transient jaundice and compensated hemolytic anemia. One of her grandchildren was also GCS deficient; he was 11 years old and had a history of neonatal jaundice. The enzyme defect was confirmed and GCS activity was found to be less than 2% of normal in the erythrocytes of both patients. The complementary DNA (cDNA) for the heavy subunit of GCS was sequenced in these patients and in several members of the family. The proband and her GCS- deficient grandson were identified as homozygous for a 473C-->T substitution, changing codon 158 from CCC for proline into CTC for leucine. Several family members with half-normal GCS activity in their erythrocytes were heterozygous for the mutation.

Aged↗
PubMed2000

[Fertility and contraception in women older than 40 years].

Women in their later reproductive years constitute a large proportion of contraceptive users. Although increasing age is accompanied by a general decline in fecundity, each individual needs to know her own risk or possibility of becoming pregnant and when it is safe to stop using contraceptives. Women over 40 need special consideration since pregnancy and childbirth is sometimes associated with higher health risks both for mother and infant. Women over 40 are more likely than younger women to have medical problems, menstrual disorders and previous experience that may affect their choice and use of contraceptive methods. Recommendations for counselling, health screening and choice of contraceptive method in women in their later reproductive years are given.

Adult↗
PubMed2000

Biosensor analysis of drug-target interactions: direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors.

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specificity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 microM concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel.

Antithrombins↗
PubMed2000

Inhibition of debrisoquine hydroxylation with quinidine in subjects with three or more functional CYP2D6 genes.

AIMS: To study whether the CYP2D6 capacity in ultrarapid metabolizers of debrisoquine due to duplication/multiduplication of a functional CYP2D6 gene, can be 'normalised' by low doses of the CYP2D6 inhibitor quinidine and whether this is dose-dependent. METHODS: Five ultrarapid metabolizers of debrisoquine with 3, 4 or 13 functional CYP2D6 genes were given single oral doses of 5, 10, 20, 40, 80 and 160 mg quinidine. Four hours after quinidine intake, 10 mg debrisoquine was given. Urine was collected for 6 h after debrisoquine administration. Debrisoquine and its 4-hydroxymetabolite were analysed by h.p.l.c. and the debrisoquine metabolic ratio (MR) was calculated. RESULTS: Without quinidine the MR in the ultrarapid metabolizers ranged between 0.01 and 0.07. A dose-effect relationship could be established for quinidine with regard to the inhibitory effect on CYP2D6 activity. To reach an MR of 1-2, subjects with 3 or 4 functional genes required a quinidine dose of about 40 mg, while the sister and brother with 13 functional genes required about 80 mg quinidine. After 160 mg quinidine, the MRs, in the subjects with 3, 3, 4, 13 and 13 functional genes, were 12.6, 10.1, 9.2, 2.4 and 2.2, respectively. CONCLUSIONS: A dose-effect relationship could be established for quinidine inhibition of CYP2D6 in ultrarapid metabolizers. The clinical use of low doses of quinidine as an inhibitor of CYP2D6 might be considered in ultrarapid metabolizers taking CYP2D6 metabolized drugs rather than giving increased doses of the drug. Normalizing the metabolic capacity of CYP2D6, by giving a low dose of quinidine, may solve the problem of 'treatment resistance' caused by ultrarapid metabolism.

Administration, Oral↗
PubMed1999

Kinetic characterization of the interaction of the Z-fragment of protein A with mouse-IgG3 in a volume in chemical space.

The kinetic rate parameters for the interaction between a single domain analogue of staphylococcal protein A (Z) and a mouse-IgG3 monoclonal antibody (MAb) were measured in Hepes buffer with different chemical additives. Five buffer ingredients (pH, NaCl, DMSO, EDTA, and KSCN) were varied simultaneously in 16 experiments following a statistical experimental plan. The 16 buffers thus spanned a volume in chemical space. A mathematical model, using data from the buffer composition, was developed and used to predict apparent kinetic parameters in five new buffers within the spanned volume. Association and dissociation parameters were measured in the new buffers, and these agreed with the predicted values, indicating that the model was valid within the spanned volume. The pattern of variation of the kinetic parameters in relation to buffer composition was different for association and dissociation, such that pH influenced both association and dissociation and NaCl influenced only dissociation. This indicated that the recognition mechanism (association) and the stability of the formed complex (dissociation) involve different binding forces, which can be further investigated by kinetic studies in systematically varied buffers.

Animals↗
PubMed1999

Early hypoxia modulates the phenotype of dopaminergic cells in rat di- and mesencephalic cell cultures and induces a higher vulnerability of non-dopaminergic neurons to a second hypoxic exposure.

To investigate long-term effects of hypoxia on a cellular level, di- and mesencephalic cell cultures were exposed to hypoxia on in vitro day 2 (incubation in culture medium, pO2 = 10-20 mmHg, 24 h) and on in vitro day 13 (incubation in an electrolyte solution, pO2 = 10-20 mmHg, 8 h). The numbers of neuron-specific enolase immuno-reactive (NSE-IR) and tyrosine hydroxylase immuno-reactive (TH-IR) neurons and the levels of dopamine, its main metabolites and the spontaneous and potassium-stimulated DA release were determined on DIV 15. Hypoxia on DIV 2 did not affect the numbers of NSE-IR and TH-IR neurons, but increased the dopamine content and dopamine release by about 100% in both di-and mesencephalic cultures. In addition, this hypoxia increased the vulnerability of non-TH-IR neurons to the second hypoxic episode applied during more advanced stages of the culture development on DIV 13. On the contrary, hypoxia exposure did not affect the vulnerability of TH-IR cells.

Animals↗
PubMed1999

Gastrin effects on isolated rat enterochromaffin-like cells following long-term hypergastrinaemia in vivo.

The enterochromaffin-like (ECL) cells play an important role in the regulation of gastric acid secretion. They respond to gastrin by a prompt increase in histamine secretion, an effect which is mediated by the CCK-(B)/gastrin receptor acting through the IP(3)/DAG pathway. In the rat, long-term treatment with acid secretion inhibitors induces hypergastrinaemia which, in turn, results in ECL cell hypertrophy and hyperplasia. The aim of the present study was to evaluate various functional parameters in acutely isolated rat ECL cells, following long-term hypergastrinaemia in vivo. Rats were treated with vehicle or a supramaximal daily dose of omeprazole for more than 10 weeks to ensure ECL cell hyperplasia. ECL cells were isolated from vehicle-treated animals and 24, 72 and 120 h after the last dose of omeprazole. The functional activity of the acutely isolated ECL cells was determined by measuring gastrin-and forskolin-induced histamine secretion. Changes in cytosolic free calcium upon gastrin stimulation were monitored by digital video imaging. ECL cells successively regained their ability to respond to gastrin following long-term hypergastrinaemia, reaching close to vehicle-treated levels 120 h after the last dose of omeprazole. In the rat, the response pattern of the ECL cells appears to normalise in parallel with the normalisation of plasma gastrin levels.

Animals↗
PubMed1999

Identification and optimization of regeneration conditions for affinity-based biosensor assays. A multivariate cocktail approach.

A general regeneration, identification, and optimization (RO) protocol for Biacore systems was developed. The RO protocol uses six multi-ingredient stock solutions that represent the six most common chemical properties employed as regeneration agents. The regeneration effect of different regeneration cocktails of these six stock solutions were tested iteratively until a satisfactory result was obtained. The RO protocol was designed with an ease-of-use and multivariate approach. The RO protocol was tested on 13 different antibody-antigen systems. For 10 of these, only the first screening session was tested. For 9 of the 13 systems, the RO-protocol screening session identified cocktails that removed more than 90% of the bound analyte in a 30 s pulse. For 5 systems, the RO protocol identified cocktails that regenerated the surface completely and that were more gentle than previously used regeneration conditions. Furthermore, the regeneration optimization results can be interpreted as a characterization of the interacting molecules. The relevance of testing cocktails was justified by the fact that at least one cocktail was significantly better than all diluted stock solutions for all tested model systems. By using the multivariate approach, the risk of missing relevant combinations of stock solutions was minimized. This resulted in an unexpected discovery of excellent properties of EDTA as an additive in regeneration cocktails containing chaotropic agents and ions in high concentration.

Biosensing Techniques↗
PubMed1999

Exposure of cryptic epitopes on transthyretin only in amyloid and in amyloidogenic mutants.

The structural requirements for generation of amyloid from the plasma protein transthyretin (TTR) are not known, although it is assumed that TTR is partly misfolded in amyloid. In a search for structural determinants important for amyloid formation, we generated a TTR mutant with high potential to form amyloid. We demonstrated that the mutant represents an intermediate in a series of conformational changes leading to amyloid. Two monoclonal antibodies were generated against this mutant; each displayed affinity to ex vivo TTR and TTR mutants with amyloidogenic folding but not to wild-type TTR or mutants exhibiting the wild-type fold. Two cryptic epitopes were mapped to a domain of TTR, where most mutations associated with amyloidosis occur and which we propose is displaced at the initial phase of amyloid formation, opening up new surfaces necessary for autoaggregation of TTR monomers. The results provide direct biochemical evidence for structural changes in an amyloidogenic intermediate of TTR.

Amino Acid Sequence↗
PubMed1999

YopH dephosphorylates Cas and Fyn-binding protein in macrophages.

The tyrosine phosphatase YopH is an essential virulence effector of pathogenic Yersinia spp. YopH, which is translocated from extracellularly located bacteria into interacting target cells, blocks phagocytosis by professional phagocytes. We show here that immunoprecipitation of YopH from lysates of J774 cells infected with Y. pseudotuberculosis expressing an inactive form of YopH resulted in co-precipitation of certain phosphotyrosine proteins. The association between the inactive YopH and phosphotyrosine proteins in the 120 kDa range was rapid and could be detected after 2 min of infection. The proteins were identified as the docking proteins Cas and Fyn-binding protein (FYB). Upon infection of J774 cells with Y. pseudotuberculosis lacking YopH expression both of these proteins became tyrosine phosphorylated. Moreover, this infection caused recruitment of Cas to peripheral focal complexes, and FYB was relocalized to areas surrounding these structures. Both Cas and FYB became dephosphorylated upon infection with Y. pseudotuberculosis expressing active YopH, and this was associated with disruption of focal complexes. With regard to the previous identification of Cas and focal complexes as targets of YopH in HeLa cells, the present study supports an important role for these targets in a general mechanism of bacterial uptake.

Bacterial Outer Membrane Proteins↗
PubMed1999

Fumonisin B1-induced DNA damage in rat liver and spleen: effects of pretreatment with coenzyme Q10, L-carnitine, alpha-tocopherol and selenium.

Active oxygen radical species are reported to cause organ damage. This study was designed to determine whether oxidative stress contributed to the initiation or progression of hepatic and splenic cell DNA damage induced by fumonisin B1 (FB1) in rats. Another aim was to investigate the protective effects of the antioxidants coenzyme Q10 (CoQ10), L-carnitine, vitamin E (alpha-tocopherol) and selenium against DNA damage in the liver and spleen of rats treated with FB1. Fasted rats were injected intravenously with a single dose of fumonisin B1 at 1.55 mg kg-1 body wt. into the tail vein. Treatment with FB1 led to splenic and hepatic DNA fragmentation in 85% of the test animals. DNA fragmentation was investigated as a critical event in toxic cell death by testing total Ca2+ in liver. FB1 administration caused total Ca2+ in liver to increase within 4 h (204% of control). Measurement of liver enzyme activities showed an increase in aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT). FB1 also markedly decreased splenic and hepatic glutathione (GSH) levels. Pretreatment with CoQ10 (30 mg CoQ10 kg-1 diet) together with L-carnitine (2.8 mg carnitine kg-1 diet), alpha-tocopherol (30 IU vitamin E kg-1 diet) and selenium (1 mg selenium as sodium selenite kg-1 diet), decreased DNA damage and the activities of Ca2+, ASAT and ALAT in the liver. On the other hand, the level of GSH was slightly increased. The CoQ10 alone did not significantly protect against toxic cell death and glutathione depletion caused by FB1. Oxidative damage caused by FB1 may be one of the underlining mechanisms of FB1-induced cell injury and DNA damage.

Animals↗
PubMed1999

Hypoxia induces differential changes of dopamine metabolism in mature and immature mesencephalic and diencephalic cell cultures.

Perinatal hypoxia is known as a high risk factor for the development of long-lasting abnormalities in dopaminergic system. The early developmental alterations of dopamine (DA) metabolism induced by hypoxia could contribute to these abnormalities. To understand the hypoxia-induced changes of intra- and extracellular dopamine levels and its main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in immature dopaminergic neurons, we compared these changes in rat mesencephalic and diencephalic cell cultures on day in vitro (DIV) 2 (immature cells), DIV 8 and DIV 13 (mature cells). Cell cultures were exposed to an oxygen-free gas mixture in a Billups chamber for 2-4 hours. Mature cell cultures responded to hypoxia with an increase of DA levels in the cells and in the medium during the first 45 min (by an average of 57 and 114% respectively). Thereafter, DA levels decreased, and returned to the baseline within the next 30 min. The cellular DA levels continued to decrease up to 15% of the baseline during 255 min hypoxia whereas the extracellular DA content stabilized at the prehypoxic levels. Immature cell cultures (DIV 2) in contrast to mature ones, were unable to maintain normal extracellular DA levels during hypoxia and showed a decrease of the cellular and extracellular levels to 50% of the prehypoxic levels. DOPAC and HVA changes mimick, however, at a lower level, the pattern of DA changes during the exposure to hypoxia. In principle, in the diencephalic cell culture similar effects of hypoxia exposure on the investigated parameters were found (studied during 0-120 min). The present study demonstrates that mature and immature dopaminergic cells differ in the regulation of the extra- and intracellular DA levels during hypoxia. In immature cells the low synthetic capacity of tyrosine hydroxylase and the deficient capacities of the transport and storage processes result in decreased extracellular DA levels. This could be an important factor for the long-term modulation of the expression of tyrosine hydroxylase and subsequent long-term behavioral and/or neurological abnormalities induced by perinatal hypoxia.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1999

Hypoxia during early developmental period induces long-term changes in the dopamine content and release in a mesencephalic cell culture.

The present study was conducted to elucidate the long-term effects of exposure to hypoxia of dopaminergic neurons during the early developmental period. Primary mesencephalic cell cultures prepared from fetal rats and containing 0.5-2% of dopaminergic neurons were exposed to hypoxia between in vitro days 1 and 6, the putative critical developmental period. Changes in the content, release and uptake of dopamine were found to depend on the degree of hypoxia and on the duration of exposure. Following moderate hypoxia (7 h, 5% O2) on two consecutive days between in vitro days 1 and 3, the cultures showed a small increase in the dopamine levels, by 16%. After severe hypoxia (0% O2/95% N2 for 24 h), during the same time window, the cellular dopamine content was elevated by 100%. Moreover, severe hypoxia produced long-lasting modulations of the dopaminergic system. On in vitro day 14, cells exhibited increased levels of 3,4-dihydroxyphenylacetic acid and homovanillic acid (by 34% and 55%, respectively), and elevations of both the spontaneous and potassium-stimulated dopamine release by 70%. The dopamine transport and metabolism of cells exposed to hypoxia between in vitro days 4 and 6 remained unchanged with regard to long-term effects. The present study provides strong evidence for the induction of long-term changes in dopaminergic cells due to hypoxia during the critical developmental period in mesencephalic culture. The developmental period capable of inducing long-lasting changes in dopamine metabolism is restricted to in vitro days 1-3.

Animals↗
PubMed1999

Dexamethasone differentially regulates cytokine transcription and translation in macrophages responding to bacteria or okadaic acid.

Many microorganisms and microbial products induce expression of pro-inflammatory cytokines such as interleukin-1 (IL-1alpha/beta) and tumour necrosis factor-alpha (TNF-alpha) in macrophages, primarily by transcriptional activation. We show here, by using mouse macrophages in primary culture, that pre-treatment with dexamethasone inhibits bacteria-induced IL-1beta expression as mRNA and cellular pro-IL-1beta in parallel, consistent with an effect primarily on transcriptional activation. In contrast, the expression of TNF-alpha mRNA was only partly inhibited despite virtually complete inhibition of TNF-alpha protein formation. Furthermore, the selective induction of primarily cell-associated 26,000 M, pro-TNF-alpha by the protein phosphatase inhibitor okadaic acid also was partly inhibited at the mRNA level by dexamethasone, whereas additional translational inhibition appeared to be lacking. This latter finding is reminiscent of earlier findings regarding signalling to activation of cytosolic phospholipase A2, which is sensitive to dexamethasone when elicited by bacteria, but not when elicited by okadaic acid. The present results raise the possibility that the inhibitory effect of dexamethasone on TNF-alpha translation, but not on transcriptional activation, is mediated by one or more okadaic acid-sensitive protein phosphatases.

Animals↗