Biomedical subjects
Publications and source records attributed to R Karlsson.
Neutral or charge-shifting mutagenesis of beta-actin at positions 3 and 4 strongly influenced the actomyosin interaction under non-rigor conditions. The polymerization behaviour and tropomyosin binding properties on the other hand remained unaffected.
A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.
The serum concentrations of osteocalcin (bone Gla protein) were followed by continuous blood sampling for 24 h in 9 healthy young women before and during treatment with oestrogen/progestogen combinations for oral contraception. There were marked fluctuations during the 24 h sampling period, values ranging from 0.5 to 10.0 ng/ml. Values displayed an apparent circadian rhythm. Daytime values were on average lower than nocturnal concentrations. During treatment with oral contraceptives there was a significant decrease in osteocalcin levels but fluctuations during the 24 h sampling period were still observed. Almost all individual values obtained at 30 min intervals were lower during treatment. For the whole group the mean osteocalcin concentration decreased by 1.4 ng/ml (p less than 0.01) during treatment. In postmenopausal women high serum levels of osteocalcin are supposed to reflect increased bone turnover secondary to enhanced bone resorption. Oestrogens are known to reduce osteocalcin levels and may reduce bone resorption. In healthy young women alternative mechanisms should be considered but the reduced osteocalcin serum levels in this short-term study indicate that oral contraceptive use may influence bone metabolism.
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Weight gain is a common side effect during oral contraceptive use. The secretion of the satiety hormone cholecystokinin was recorded for 24 hours in women before and during treatment with oral contraceptives. During treatment serum profiles of cholecystokinin were clearly suppressed, which might be related to increased appetite and weight gain.
The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from young women was determined by polymerase chain reaction (PCR) by using general primer pairs localized within the L1 region. With a one-step general PCR, 5.9% (35 of 590) of young women in a population-based study were found to contain HPV DNA. The proportion of HPV-positive women increased with age, from 1.4% (1 of 69) among women aged 19 years to 9.2% (13 of 142) among women aged 25 years. Among the cervical scrapes from women with normal cytology, 5.6% (30 of 539) harbored HPV DNA. A total of 5 of 19 (26.3%) of the women with pathological signs were positive for HPV DNA. By a two-step PCR, using nested general primers, 20.3% (118 of 581) of all women were shown to contain HPV DNA. The proportion of HPV-positive women also increased with age, from 17.4% (12 of 69) among women aged 19 years to 31.9% (43 of 135) among women aged 25 years, when the two-step PCR was used. Some 19.2% (102 of 530) of cervical scrapes from women with normal cytology contained HPV DNA. Among the women with pathological signs, 16 of 19 (84.2%) were positive for HPV DNA. The HPV DNA-positive specimens were demonstrated to contain HPV type 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 55, or 56. The most prevalent HPV types were 6 (2.0%) and 16 (2.7%). More than one type was found in 16 specimens. Sixty HPV-positive samples could not be typed.
The interpretation of previous data on osteocalcin during pregnancy has been complicated by the fact that serum levels, at least in nonpregnant women, display a significant circadian variation. In the present study, serum concentrations of osteocalcin were recorded for 24 h in 12 individual women. In 4 nonpregnant women, there were striking diurnal fluctuations during the sampling period, with values ranging from 0.5 to 4.0 ng/ml. One woman who was investigated in the 11th week of pregnancy showed similar fluctuations in osteocalcin concentrations whereas in the 15th and 17th week values were much lower with minor fluctuations. In 4 women investigated during late pregnancy (weeks 34-38), osteocalcin serum levels were extremely low (range 0.2-0.4 ng/ml), often below the detection limit of the assay, and there was no diurnal variation. Also, in 1 lactating woman, osteocalcin serum levels were low (0.3-0.8 ng/ml) and stable. Low osteocalcin values during pregnancy may indicate a reduced bone turnover possibly mediated via an altered estrogen and growth hormone secretion.
An enzyme linked immunosorbent assay for rat angiotensinogen was developed based on one monoclonal antibody with high affinity for angiotensinogen and des-angiotensin 1-angiotensinogen and rabbit polyclonal antibodies for angiotensinogen was developed. Serum levels of angiotensinogen were lower in female than in male rats but increased significantly after hypophysectomy. Estrogen substitution after hypophysectomy had no further stimulatory or inhibitory influence. In hypophysectomized animals continuous and intermittent growth hormone administration had clearly different effects. The results indicate that the sexually dimorphic secretion of growth hormone is involved in the regulation of circulating angiotensinogen concentrations in the rat.
An automated biosensor system for measuring molecular interactions has been used to study the kinetics of monoclonal antibody-antigen reactions. The system combines a microfluidic unit in contact with a sensor surface for surface plasmon resonance detection. The specificity of the surface is determined by the operator. Antibody or antigen is immobilised in a dextran matrix attached to the sensor surface. The interaction of matrix bound antibody or antigen with the corresponding partner in solution is monitored in real time. None of the interacting molecules needs to be labelled and it is not necessary to determine the concentration of the the matrix bound component in advance. Two systems were studied: matrix bound monoclonal antibodies (MAbs) interacting with HIV-1 core protein p24 and immobilised aminotheophylline reacting with MAbs. Control of the amount of immobilised ligand and reusable sensor surfaces permits the comparison of different MAbs reacting with antigen under almost identical conditions. Differences in affinity and reaction rates are immediately apparent. The calculated association rate constants for p24 MAbs ranged from 3 x 10(4) - 7.4 x 10(5) M-1 s-1 and for theophylline MAbs association rate constants as high as 1 x 10(6) M-1 s-1 were encountered. The calculated dissociation rate constants were in the region 2 x 10(-4) s-1 to 2 x 10(-2) s-1.
Three N-terminal double mutants of beta-actin expressed in the yeast Saccharomyces cerevisiae have been characterized with respect to DNase-I interaction, N-terminal post-translational modification, polymerizability and myosin subfragment-1 binding. The results strongly support earlier suggestions that the acidic residues at the N-terminus of actin are part of the myosin-binding site, while they seem to be of no importance for the other aspects of actin biochemistry tested. The suitability of this expression system for production of recombinant actin in general is discussed.
The yeast Saccharomyces cerevisiae was used to express human profilin cDNA. The recombinant protein, isolated by affinity chromatography on poly(L-proline)-Sepharose followed by ion exchange chromatography, associates with non-muscle actin and phosphatidylinositol-(4,5)-bisphosphate as authentic profilin.
Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.
We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.
The relationship between the rate of pressure applied over the masseter muscle and the pressure pain threshold was studied with the aid of an algometer (pressure threshold meter, PTM) connected to a stopwatch. Twenty consecutive patients with various degrees of craniomandibular dysfunction were included in the study. In 10 of the patients, the belly of the right superficial masseter muscle was recorded with the algometer 10 times using different pressure rates arbitrarily and unsystematically ranging from low to high values. The correlation between pressure rate and the PTM value was high. An increased pressure rate increased the PTM value (mean regression coefficient b = 0.70). A PTM value range of 0.5 kg/cm2 was found to correspond to a pressure rate range of 0.50 kg/cm2/s. In the remaining 10 patients, repeated recordings of the same site with a constant pressure rate did not influence the PTM value significantly.
A 3-year, double-blind, randomized caries trial was conducted to evaluate the relative anticaries efficacy of four sodium fluoride dentifrices containing 250 ppm fluoride, 1,000 ppm fluoride in combination with 1% disodium 1-hydroxyethylidene-1.1-bisphosphonate (HEBP), and 1,000 ppm fluoride in combination with 1% disodium azacycloheptylidene-2.2-bisphosphonate (AHBP). As a positive control, a monofluorophosphate dentifrice (1,000 ppm fluoride) was used. At outset 1,161 Icelandic children, 11 and 12 years of age, were randomly assigned to one of the five treatment groups and 1,035 subjects completed the trial. After 3 years of unsupervised brushing, the dentifrice containing 250 ppm fluoride was significantly less effective in controlling the caries increment. The combination of sodium fluoride and AHBP was significantly more effective than the positive control.
Serum profiles of growth hormone (GH) were recorded for 24 h in 9 young healthy women before and during treatment with monophasic, combined contraceptives. During treatment, there was a change in GH secretion towards a pattern of smaller peaks at a higher frequency. The mean peak amplitude decreased from 11.2 to 7.3 mU/l, the mean peak area from 24.1 to 14.1 mU/l, and the mean interpeak interval from 4.3 to 3.0 h. The biological impact of altered GH secretion is well-known from animal experiments. A sex-steroid-induced change in GH secretion may be relevant for certain metabolic effects of oral contraceptives.
Actin interacts with a number of so-called actin-binding proteins which participate at various stages of the cell motility process such as regulation of filament formation, assembly and disassembly of filaments, force generation and depolymerization. Gene technology makes a precise mapping of the interacting surfaces on the actin molecules possible by studying specifically designed actin mutants expressed in a suitable organism. In addition, the production of engineered actin will become increasingly important when the three-dimensional structure of actin is determined. Chicken beta-actin can be produced in large quantities in Escherichia coli but such actin shows only a limited biological activity and thus seems to be of minor interest in future studies of structure-function relationships of this molecule. To circumvent the problem of a denatured bacterial protein, the yeast Saccharomyces cerevisiae was chosen as an alternative organism to express actin. This paper describes the expression, isolation and characterization of the yeast-produced chicken beta-actin. From a 12-liter culture of yeast cells, 500 micrograms of polymerizable beta-actin was isolated.
Skeletal muscle troponin C (TNC) is structured into two separate domains linked by a nine-turn alpha-helix (D/E helix). It has been demonstrated that calcium binding to the regulatory sites within the N-terminal domain induces conformational changes in the C-terminal domain of isolated TNC. Since the only contact between the two domains is the long D/E helix, the transfer of information must involve conformational changes within this helix. The center of the helix is occupied by a glycine (Gly-92). A postulated mechanism for allowing interdomain interaction involves a conformational change of the D/E helix around Gly-92 (Herzberg, O., and James, M. N. G. (1985) Nature 312, 653-659). We tested this hypothesis using site-directed mutants of troponin C. Two separate mutants containing an alanine and a proline replacing Gly-92 were constructed and compared with wild type TNC. Calcium binding studies showed no significant differences among the TNC species. The different TNC were assembled into thin filaments and used to assay the calcium regulation of actin-activated ATPase of myosin. All TNC species were able to mediate the calcium regulation of ATPase. Under the conditions used for the assays, no differences were detected among the TNC species. These results show that Gly-92 is not essential for the proper interaction of the calcium regulatory sites with the other components of the thin filament, and therefore exclude a large rotation around Gly-92 as the mechanism of information transfer between the two domains of troponin C.