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Biomedical subjects

R Karlsson

Publications and source records attributed to R Karlsson.

At least 19 recordsLinked to original sources
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↗
PubMed1995

Identification of the trapped calcium in the gelsolin segment 1-actin complex: implications for the role of calcium in the control of gelsolin activity.

The X-ray structure of the complex of actin with gelsolin segment 1 revealed the presence of two calcium ions, one bound at an intramolecular site within segment 1 and the other bridging the segment directly to actin. Although earlier calcium binding studies at pH 8.0 revealed only a single calcium trapped in the complex (and also in the binary gelsolin-actin complex), it is here shown that two calcium ions are bound under the conditions of crystallization at physiological pH. Mutation of acidic residues in either actin or segment 1 involved in ligation of the intermolecular calcium ion resulted in loss of one of the bound calcium ions at pH < 7, but not at pH 8. Thus the calcium ion trapped in the segment 1-actin complex is that located at the intramolecular site. The implications of this for gelsolin function are discussed.

Actins↗
PubMed1995

Human papillomavirus infection is transient in young women: a population-based cohort study.

The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from a cohort of 276 young women was determined by a general two-step polymerase chain reaction. HPV infection fluctuated among young women during a 2-year interval. The total prevalence of HPV infection decreased from 21% to 8.3%. The most prevalent HPV types at enrollment were HPV-16 (3.3%) and HPV-6 (2.9%). At follow-up, the most common type was HPV-16 (2.9%), while no HPV-6 was detected. In 2 women only, the same HPV type persisted. Regression of HPV infection was found in 80% of the women. A new HPV type-specific infection was detected in 7.2% of the women and was independently associated with a new sex partner or an abnormal smear since enrollment.

Adult↗
PubMed1995

The silent suffering women--a population based study on the association between reported symptoms and past and present infections of the lower genital tract.

OBJECTIVES: To assess the prevalence of lower genital tract symptoms and the association between reported symptoms and past and present signs of sexually transmitted diseases (STD) in young women. DESIGN: All women belonging to the 19-, 21-, 23- and 25-year age cohorts and living in the catchment area of the community health centre, were invited by mail to take part in a population-based study. The participants answered a structured questionnaire and a gynaecologic examination was performed. Samples for wet smear, cervical Pap smear, HPV DNA determination and Chlamydia trachomatis culture were taken at the gynaecologic examination. The presence of genital warts was noted. A blood sample was analysed for antibodies against C trachomatis and HSV-2. SETTING: The community health care centre was located in Umeå, a city in Northern Sweden. RESULTS: Of the 886 women who were eligible, 611 (70%) participated in the investigation. One out of four women reported symptoms from the lower genital tract. The most commonly reported symptoms were itching, followed by discharge, and soreness. The most commonly reported STD was C trachomatis (15%). The most prevalent present STD was HPV infection (20%) whereas C trachomatis infection could be isolated from 2.7% of the women. Antibodies against C trachomatis and HSV-2 were present among 22% and 6% of the women, respectively. There was a significant correlation between the women's complaint of vaginal discharge and previous C trachomatis infection, lack of lactobacilli and presence of leucocytosis in wet smear. CONCLUSIONS: We have in a population-based study of young healthy women found that one out of four women had some kind of lower genital tract complaint. Itching was the most commonly reported symptom and was associated with pseudohyphae and acetowhite patches. Reported vaginal discharge and soreness were associated with the history of a past C trachomatis infection and signs of a disturbed vaginal flora.

Adult↗
PubMed1994

Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors.

With surface plasmon resonance detection it is possible to measure the binding kinetics between a macromolecule in solution and a receptor immobilized on a sensor surface. The detector response is proportional to the mass of the analyte that binds to the surface, and therefore, a direct observation of a low-molecular-weight (lmw) analyte ( < 5000 Da) interacting with its immobilized binding partner is normally not possible. I describe here a competitive approach in which a lmw analyte and a high-molecular-weight analyte react at the same time with the immobilized receptor. Using this approach it is possible to extend kinetic analysis to lmw analyte-receptor interactions. A qualitative analysis allows rapid affinity ranking of different lmw analytes interacting with the same receptor, and a quantitative analysis of binding data allows the calculation of rate constants for the lmw analyte-receptor interaction. The competitive kinetics approach may therefore be used as an alternative to other affinity techniques for the characterization of lmw ligands, for identification of inhibitors, and for drug screening.

Amino Acid Sequence↗
PubMed1994

Working on a moving surface--a biomechanical analysis of musculo-skeletal load due to ship motions in combination with work.

Ship motions are recognized by fishermen as a cause of high musculo-skeletal load. In the present study, the motions of a Swedish trawler at sea were registered over three degrees of freedom. Simultaneously, the working postures of a fisherman on board were registered in the sagittal plane during five different working situations: (1) standing erect in still conditions; (2) holding a load of 21 kg in still conditions; (3) standing erect during motion of the ship; (4) standing during motion of the ship, holding a load of 21 kg; and (5) repeatedly lifting and lowering a 21 kg load during motion of the ship. All registrations were sampled using a computer where data were processed in a two-dimensional, dynamic biomechanics model, developed for this particular purpose. Vertical and horizontal forces as well as moments were calculated for seven major joint systems of the body, as was compression at the L4/L5 vertebral level. In situation (3), ship motions were mainly counteracted by motions in the lower extremity and lumbar back, thus inducing increased strain in these parts of the body; other parts of the body were little affected. Holding a load considerably increased the load on most joints. Lifting the load further increased the musculo-skeletal strain and also increased the range of moments in each joint as well as the range of lumbar compression. Moment at the C7 vertebral level was relatively unaffected by ship motions and by handling external loads.

Biomechanical Phenomena↗