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Biomedical subjects

R Karlsson

Publications and source records attributed to R Karlsson.

At least 37 records · Page 2Linked to original sources
PubMed1999

Timing of peak serum cortisol values in preterm infants in low-dose and the standard ACTH tests.

The low-dose ACTH test seems to reveal mild cases of adrenal insufficiency and is beginning to be preferred over the standard ACTH test in the evaluation of adrenal suppression both in infants and in adults. The concentration-time profile of plasma cortisol in infants after a low ACTH dose is obscure. In this crossover study, we compared timing of the peak values in the low-dose and the standard ACTH stimulation tests in preterm and full-term infants. We performed the standard ACTH tests (250 microg/1.73 m2) and the low-dose ACTH tests (1 microg/1.73 m2) on 10 infants (26-40 wk gestational age) and measured serum cortisol concentration at 0, 30, 40, 60, and 120 min by RIA. Nine of the infants had received postnatal glucocorticoid treatment, and most of them had also been treated with dexamethasone antenatally. In the low-dose test, the peak values occurred at 30 or 40 min in 9/10 patients. In the standard-dose test, the peak values occurred at 60 or 120 min in 8/10 patients. These results are comparable with those from adults. According to this study, blood samples for the low-dose ACTH test in infants should be taken before dosing and between 30 and 40 min after dosing.

Adult↗
PubMed1998

The role of profilin in actin polymerization and nucleotide exchange.

Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.

Actins↗
PubMed1998

Thermodynamic analysis of protein interactions with biosensor technology.

A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.

Animals↗
PubMed1998

Antenatal dexamethasone treatment decreases plasma catecholamine levels in preterm infants.

Antenatal corticosteroid therapy (ACT) has many beneficial effects on preterm infants. The cellular mechanisms of action of ACT include beta-adrenergic receptor-mediated cAMP generation. This study investigated the effects of ACT on sympathoadrenal mechanisms during immediate postnatal adaptation of preterm infants. Plasma epinephrine, norepinephrine, 3,4-dihydroxyphenylglycol, and cAMP were measured within 12 h after birth in 103 preterm infants (gestational age 24-36 wk), who were divided into two groups (non-ACT and ACT group) according to whether the mother had received dexamethasone treatment. Infants in the ACT group had significantly lower concentrations of plasma catecholamines than infants in the non-ACT group; plasma epinephrine was 38% lower, and plasma norepinephrine was 20-40% lower in the ACT group, depending on gestational age (r = -0.37 in the non-ACT group and r = -0.28 in the ACT group, p < 0.05). Plasma cAMP concentrations were similar in the two groups. Antihypertensive treatment of the mother was associated with low plasma cAMP (p < 0.001), whereas tocolytic treatment was associated with high plasma cAMP (p = 0.001) of the infant. The results indicate that ACT attenuates the birth-related increase in plasma catecholamines. Still, plasma cAMP levels remain high, which suggests enhanced beta-adrenoceptor signaling after ACT.

Antihypertensive Agents↗
PubMed1997

Isolation and characterization of two mutants of human profilin I that do not bind poly(L-proline).

A simple procedure for the isolation of profilin mutants having a reduced capacity to bind poly(L-proline) is used to isolate two mutants of human profilin I, W3N and H133S. Binding of the mutants to poly(L-proline), actin, and phosphatidylinositol (4,5)-bisphosphate (PIP2) was studied. Both mutations abolished the poly(L-proline)-binding activity of profilin. This suggests that the arrangement of the N- and C-terminal helices forming the poly(L-proline)-binding site depends on the stabilizing interaction between W3 and W31 in the underlying beta-strand, and that the H133S mutation in the C-terminal helix also must have distorted the arrangement of the terminal helices. Both mutations caused a reduced affinity for actin, with the W3N replacement having the most pronounced effect. This shows that structural changes in the poly(L-proline)-binding region of profilin can affect the distantly located actin-binding site. Thus, ligands influencing the structure of the poly(L-proline)-binding site may regulate actin polymerization through profilin. This is consonant with the finding that PIP2, which changes the tryptophan fluorescence in wild-type profilin and dissociates the profilin:actin complex in vitro, binds more strongly to the W3N mutant profilin. Thus, the poly(L-proline)-binding surface represents a crucial regulatory site of profilin function.

Contractile Proteins↗
PubMed1997

Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo.

We are investigating structure-function relationships in profilin and actin by site-specific mutagenesis using a yeast, Saccharomyces cerevisiae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.

3T3 Cells↗
PubMed1997

Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors.

The reaction between antibody immobilized to surfaces, with and without a dextran matrix, and antigen in solution was studied using surface plasmon resonance detection. The use of a reference surface made it possible to identify conditions where a response related to changes in matrix conformation could be ignored. It was possible therefore to improve data quality by separating signals related to binding events from signals due to differences in refractive index between sample and running buffer. When antigen was injected over antibody immobilized to surfaces with and without dextran matrix the binding curves were virtually superimposable. Consequently, no binding artifacts associated with the dextran matrix were observed. Sets of binding curves obtained with different antigen concentrations were analyzed using numerical integration of differential rate equations and global fitting. When data was inconsistent with a one to one reaction it was possible to obtain good fits to an entire data set assuming several other reaction schemes including parallel, competitive and two-state reactions. Thus data analysis alone was not sufficient to discriminate between different reaction schemes. In contrast several reaction schemes could be ruled out with simple experiments; the duration of antigen injection, and reanalysis of antigen recovered in fractions from the antibody surface. In view of these findings experimental design appears to be the key to successful interaction analysis.

Animals↗
PubMed1997

Acetowhitening of the cervix and vulva as a predictor of subclinical human papillomavirus infection: sensitivity and specificity in a population-based study.

OBJECTIVE: To evaluate acetowhite changes of the cervix and vulva as a predictor of human papillomavirus (HPV) infection. METHODS: In this population-based study all women aged 19, 21, 23, and 25 years and registered as living in a primary health care area within the city of Umeå, Sweden were eligible for inclusion. Each participant underwent a gynecologic examination with sampling of epithelial cells for HPV-DNA detection and Papanicolaou smear. Colposcopy was performed 5 minutes after application of 5% acetic acid. A two-step polymerase chain reaction (PCR) technique was employed for HPV-DNA detection. RESULTS: Colposcopy and sampling of epithelial cells could be performed in 535 women. The sensitivity of detection of HPV infection by the acetowhitening of the cervix was 22% (95% confidence interval [CI] 18%, 26%). The specificity of detection of HPV infection by the acetowhitening of the cervix was 90% (95% CI 87%, 93%). The sensitivity of detection of HPV infection by cytology was 13% (95% CI 10%, 16%), and the specificity was 99% (95% CI 98%, 100%). The combination of acetowhitening and cytology did not improve the diagnostic value. CONCLUSION: Acetowhitening of the cervix and vulva has low sensitivity as a predictor of HPV infections as determined by PCR.

Acetic Acid↗
PubMed1997

The associations between risk behaviour and reported history of sexually transmitted diseases, among young women: a population-based study.

The aim of this study was to determine the associations between risk behaviour and women's reported sexually transmitted diseases (STDs). All the women aged 19, 21, 23 and 25, residing in a specified housing area, were invited to answer a questionnaire regarding their sexual behaviour, smoking and alcohol consumption and previous history of STD. Of the 611 women participating, one out of 4 women had a history of at least one STD. In an univariate analysis, self-reported STD was found to be related to age, having more than 4 lifetime sexual partners, having practised intercourse at first date, inconsistent use of condoms, alcohol consumption of more than 3 bottles of wine per month and smoking. These factors were, however, not independent of each other and when subjected to a multivariate logistic regression analysis 2 factors, i.e. the lifetime number of sexual partners (more than 4 partners vs one; OR 7.94, (3.41-18.50)) and coitus on first date (practised more than once vs never, OR 2.99 (1.55-5.78)) emerged as independently associated with a previous STD.

Adult↗
PubMed1996

Potentiation by estramustine of the cytotoxic effect of vinblastine and doxorubicin in prostatic tumor cells.

The effects of combining estramustine (EM) with vinblastine (VLB) or doxorubicin (DOX) on cellular uptake, cellular retention and cell survival were investigated in Dunning hormone-insensitive rat prostate AT-1 tumor cells and DU-145 human prostatic tumor cells. Accumulation of VLB and DOX by AT-1 cells was less than one-half of that in DU-145 cells. Inclusion of EM or estromustine considerably increased uptake of both VLB and DOX in AT-1 cells but not in DU-145 cells. Verapamil and tamoxifen also potentiated VLB uptake in AT-1 cells. A combination of VLB and EM resulted in a considerable synergistic effect on both cytotoxicity and cellular retention of VLB. The presence of P-glycoprotein (Pgp) in AT-1 cells could be demonstrated by both Western blots and immunocytochemical detection. Photoaffinity labeling of Pgp by [3H]-azidopine was clearly inhibited by VLB, verapamil and EM. Our data strongly support the argument for a combination of EM with not only VLB but also DOX to improve the therapeutic index in patients with prostate cancer.

Animals↗
PubMed1995

Binding of autoreactive mouse anti-type II collagen antibodies derived from the primary and the secondary immune response investigated with the biosensor technique.

The reactivity of autoantibodies to type II collagen, secreted by B cells isolated from the primary and the secondary immune response to rat type II collagen in DBA/1 mice, was investigated using BIAcore 2000 instrumentation. Assays were performed on both collagen and antibody surfaces. These assays demonstrated a 100-fold difference in affinity between primary and secondary immune response antibodies. The difference in affinity was almost entirely due to differences in the dissociation rate constant. Somatic mutations in secondary clones were in one case associated with a 3-4-fold difference in affinity and in another case appeared to be without any effect on the binding activity.

Animals↗
PubMed1995

The structure of OmpF porin in a tetragonal crystal form.

BACKGROUND: OmpF porin is a trimeric integral membrane protein responsible for the passive transport of small hydrophilic molecules, such as nutrients and waste products, across the outer membrane of Escherichia coli. Very few membrane proteins have been crystallized in three dimensions, yet this stable protein can be obtained in several crystal forms. Comparison of the structures of the same membrane protein in two different packing environments is of major interest, because it allows us to explore the integrity of the structure outside the natural membrane environment. RESULTS: The structure of OmpF porin in a tetragonal crystal form with two trimers per asymmetric unit has been determined at 3.2 A resolution and compared with that obtained previously in a trigonal crystal form. The lattice contacts involve only polar atoms, whereas extensive hydrophobic protein-protein interactions were found in the trigonal lattice. The trimer structure is virtually identical in both. CONCLUSIONS: Our comparison reveals that the overall structure of OmpF is not influenced by crystal lattice constraints and, thus, presumably bears close resemblance to the in vivo structure. The tetragonal crystal structure has provided the starting model for the phasing of neutron diffraction data obtained from this crystal form, as described in an accompanying article.

Amino Acid Sequence↗
PubMed1995

Surface plasmon resonance detection and multispot sensing for direct monitoring of interactions involving low-molecular-weight analytes and for determination of low affinities.

Surface plasmon resonance detection allows direct observation of molecular interactions between an analyte in solution and its immobilized binding partner. The use of simultaneous monitoring of interaction events on multiple sensing surfaces, with varying amounts of immobilized receptor, for detection of low-molecular-weight analytes and for determination of low affinities was investigated. Using multispot sensing and BIAcore 2000 instrumentation, analytes as small as 180 Da were detected and affinities in the 50 microM range could be determined. The use of multispot sensing thus extends direct interaction analysis to include low-molecular-weight analytes and low-affinity interactions.

Amino Acid Sequence↗
PubMed1995

Direct and competitive kinetic analysis of the interaction between human IgG1 and a one domain analogue of protein A.

Direct and competitive kinetic analysis of the binding between a one domain analogue of protein A, and mutants thereof, to immobilised hIgG1 was compared using a biosensor system based on surface plasmon resonance detection. Rate constants determined from both assays were almost identical. The experiments demonstrate that competitive kinetic analysis can be used in combination with biosensor technology, and indicate that competitive kinetic analysis may extend the use of the technology to include low molecular weight analytes.

Allergy and Immunology↗