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K Carlsson

Publications and source records attributed to K Carlsson.

At least 19 recordsLinked to original sources
PubMed2001

Probing inhibitor-induced conformational changes along the interface between tissue factor and factor VIIa.

Upon injury of a blood vessel, activated factor VII (FVIIa) forms a high-affinity complex with its allosteric regulator, tissue factor (TF), and initiates blood clotting. Active site-inhibited factor VIIa (FVIIai) binds to TF with even higher affinity. We compared the interactions of FVIIai and FVIIa with soluble TF (sTF). Six residues in sTF were individually selected for mutagenesis and site-directed labeling. The residues are distributed along the extensive binding interface, and were chosen because they are known to interact with the different domains of FVIIa. Fluorescent and spin probes were attached to engineered Cys residues to monitor local changes in hydrophobicity, accessibility, and rigidity in the sTF--FVIIa complex upon occupation of the active site of FVIIa. The results show that inhibition of FVIIa caused the structures around the positions in sTF that interact with the protease domain of FVIIa to become more rigid and less accessible to solvent. Thus, the presence of an active site inhibitor renders the interface in this region less flexible and more compact, whereas the interface between sTF and the light chain of FVIIa is unaffected by active site occupancy.

Amino Acid Chloromethyl Ketones↗
PubMed2001

alpha-Actin: disposition, quantities, and estimated effects on lung recoil and compliance.

We have investigated the basis and implications of pneumoconstriction by measuring disposition and quantities of alpha-smooth muscle actin in rat and guinea pig lungs and modeling its effects on lung recoil and compliance. A robust marker of contractility, alpha-smooth muscle actin appears in smooth muscle or myofibroblast-like cells in pleura, airways, blood vessels, and alveolar ductal tissues. In each site, we measured its transected area by immunofluorescent staining and frequency-modulated scanning confocal microscopy. We incorporated these data in a model of the parenchyma consisting of an extensive elastic network with embedded contractile structures. We conclude that contraction at any one of these sites alone can decrease parenchymal compliance by 20-30% during tidal breathing. This is due mostly to the stiffness of activated contractile elements undergoing passive cycling; constant muscle tension would have little effect. The magnitude of the effect corresponds with known responses of the lung to hypocapnia, consistent with a homeostatic function in which gas exchange is defended by redistributing ventilation away from overventilated units.

Actins↗
PubMed2000

Characterization of probe binding and comparison of its influence on fluorescence lifetime of two pH-sensitive benzo[c]xanthene dyes using intensity-modulated multiple-wavelength scanning technique.

Quantitative pH imaging using the carboxy seminaphthofluorescein dyes SNAFL-1 and SNAFL-2 can be performed by measurement of intensity ratios or fluorescence lifetimes. However, there is a controversy as to whether the latter method has the practical advantage of a straightforward pH calibration in buffers compared to a cumbersome and time-consuming procedure in cells. In this study we have undertaken a systematic study of the potential factors influencing the fluorescence lifetime of the probes at different pH using confocal microscopy. In vitro results demonstrate that factors such as lipid and protein concentrations have a substantial influence on pH measurements based on fluorescence lifetime. The pH could be overestimated by more than 2 pH units. Studies in permeabilized COS-7 cells demonstrate the same trends as observed in the in vitro studies.

Animals↗
PubMed2000

Kinetic properties of missense mutations in patients with glutathione synthetase deficiency.

Patients with hereditary glutathione synthetase (GS) (EC 6.3.2.3) deficiency present with variable clinical pictures, presumably related to the nature of the mutations involved. In order to elucidate the relationship between genotype, enzyme function and clinical phenotype, we have characterized enzyme kinetic parameters of missense mutations R125C, R267W, R330C and G464V from patients with GS deficiency. One of the mutations predominantly affected the K(m) value, with decreased affinity for glycine, two mutations influenced both K(m) and V(max) values, and one mutation reduced the stability of the enzyme. This characterization agrees well with predictions based on the recently reported crystal structure of human GS. Thus our data indicate that different mutations can affect the catalytic capacity of GS by decreasing substrate affinity, maximal velocity or enzyme stability.

Chromatography, Agarose↗
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

Confocal pH imaging of microscopic specimens using fluorescence lifetimes and phase fluorometry: influence of parameter choice on system performance.

We investigate the performance of confocal pH imaging when using phase fluorometry and fluorophores with pH-dependent lifetimes. In these experiments, the specimen is illuminated by a laser beam, whose intensity is sinusoidally modulated. The lifetime-dependent phase shift in the fluorescent signal is detected by a lock-in amplifier, and converted into a pH value through a calibration procedure. A theoretical investigation is made of how the different system parameters will influence the results concerning sensitivity and noise. Experiments carried out with the fluorophore SNAFL-2 support these theoretical predictions. It is found that, under realistic experimental conditions, we can expect a pH change of 0.1 units to be easily detected in an 8-bit digital image. However, the pixel-to-pixel root mean square noise is often of the order of one pH unit. This comparatively high level of noise has its origin in photon quantum noise. pH measurements on living cells show a systematic deviation from expected values. This discrepancy appears to be the result of fluorophore interaction with various cell constituents, and is the subject of further investigation.

Animals↗
PubMed2000

Tickling expectations: neural processing in anticipation of a sensory stimulus.

Predictions of the near future can optimize the accuracy and speed of sensory processing as well as of behavioral responses. Previous experience and contextual cues are essential elements in the generation of a subjective prediction. Using a blocked fMRI paradigm, we investigated the pattern of neural activation in anticipation of a sensory stimulus and during the processing of the somatosensory stimulus itself. Tickling was chosen as the somatosensory stimulus rather than simple touch in order to increase the probability to get a high degree of anticipation. The location and nature of the stimulus were well defined to the subject. The state of anticipation was initiated by attributing an uncertainty regarding the time of stimulus onset. The network of activation and deactivation during anticipation of the expected stimulus was similar to that engaged during the actual sensory stimulation. The areas that were activated during both states included the contralateral primary sensory cortex, bilateral areas in the inferior parietal lobules, the putative area SII, the right anterior cingulate cortex and areas in the right prefrontal cortex. Similarly, common decreases were observed in areas of sensorimotor cortex located outside the area representing the target of stimulus, i.e., areas that process information which is irrelevant to the attended process. The overlapping pattern of change, during the somatosensory stimulation and the anticipation, furthers the idea that predictions are subserved by a neuronal network similar to that which subserves the processing of actual sensory input. Moreover, this study indicates that activation of primary somatosensory cortex can be obtained without intra-modal sensory input. These findings suggest that anticipation may invoke a tonic top-down regulation of neural activity.

Adult↗
PubMed1999

Behavioral and endocrine adaptation, and up-regulation of NPY expression in rat amygdala following repeated restraint stress.

A single 1 h restraint increases experimental anxiety in the elevated plus-maze through actions within the amygdala, while intra-amygdala administration of neuropeptide Y (NPY) has the opposite effect. Endogenous amygdala NPY expression is suppressed by single restraint, providing a possible mechanism for the anxiety-promoting action of this stressor. Here, we examined whether repeated stressor exposure might lead to an adaptation (habituation or sensitization) with regard to plus-maze behavior and glucocorticoid response, and whether this might be accompanied by altered effects of the stressor on NPY expression. Following repeated restraint (1 h/day, 9-10 days), neither an anxiogenic-like effect of the stressor nor a glucocorticoid response were present. This behavioral and endocrine adaptation was accompanied by an up-regulation of prepro-NPY mRNA and NPY peptide in amygdala but not in hypothalamic or cortical extracts, an effect opposite to that previously seen after a single restraint session. Thus, an up-regulation of NPY expression in the amygdala complex may be an adaptive mechanism recruited to cope with a repeated stressor.

Adaptation, Physiological↗
PubMed1999

Neuropeptides, nitric oxide synthase and GAP-43 in B4-binding and RT97 immunoreactive primary sensory neurons: normal distribution pattern and changes after peripheral nerve transection and aging.

We have here sought to cross-correlate the expression of immunoreactivities for several neuropeptides, nitric oxide synthase (NOS) and the growth associated protein GAP-43 in subpopulations of dorsal root ganglion (DRG) neurons tagged by the selective markers isolectin B4 and the neurofilament antibody RT97, selective for, respectively, subpopulations of small and large DRG neurons. By use of double- and triple-labeling immunohistochemistry, non-manipulated and sciatic nerve transected young adult rats as well as aged (30-months-old) rats were examined using a confocal microscope equipped with enhanced spectral separation. In young adult rats, the DRG neuron profiles could be divided into three subpopulations (B4 binding (B4+) approximately 50%; RT97-immunoreactive (RT97+) approximately 35%; B4-/RT97- approximately 15%). Calcitonin gene-related peptide (CGRP) is expressed in all three subpopulations. Galanin message-associated peptide (GMAP) colocalize with CGRP (100%) but is not expressed in RT97+ profiles. NOS is present in the RT97- subpopulations and frequently colocalize with CGRP (92%). GAP-43 is expressed in all three DRG subpopulations and colocalize with CGRP (88%), GMAP (38%) and/or NOS (22%). Only very small differences were seen among the young adult rats, implicating that the size of respective subpopulation as well as the expression pattern for neuropeptides, NOS and GAP-43 are fairly stable. Sciatic nerve transection reduced B4-binding but not RT97-like immunoreactivity. Distinct changes in the expression of neuropeptides, NOS and GAP-43 were evident in the DRG subpopulations and, furthermore, the regulatory changes were very similar among the lesioned animals. The relative size of the DRG subpopulations was unaffected by aging, while the expression of neuropeptides was altered showing similarities with the changes induced by axotomy in young adult rats.

Aging↗
PubMed1998

HLA-DM is expressed on the cell surface and colocalizes with HLA-DR and invariant chain in human Langerhans cells.

The subcellular distribution patterns of molecules involved in the process of antigen loading [HLA-DR, HLA-DM, and the cytoplasmic and luminal parts of the invariant chain (Ii, CD74)] were investigated in Langerhans cells (LC), both qualitatively and quantitatively. The analysis was performed by immunofluorescence labelling of acetone-fixed vertical cryostat sections from normal human skin specimens and subsequent examination using confocal laser scanning microscopy (CSLM). The intensity-modulated multiple-wavelength scanning (IMS) technique was used to enhance channel separation when scanning dual-labelled specimens. The mean (n = 9) relative epidermal volumes of reactivity were: HLA-DR 8%+/-3%, HLA-DM 6%+/-2%, luminal Ii 6%+/-2%, and cytoplasmic Ii 4%+/-1%. The difference between HLA-DR and the other epitopes was significant at the P<0.001 level. All molecule combinations, except the combination of HLA-DM and luminal Ii (which was not studied), were to various extents colocalized. Experiments performed on unfixed epidermal sheets showed that HLA-DM is present on the cell surface of LC, suggesting that HLA-DM may interact with HLA-DR on the surface to induce peptide loading.

Adult↗
PubMed1998

Excimer laser surgery for keratoconus.

PURPOSE: To determine whether there is increased risk associated with excimer laser surgery of primary keratoconus. SETTING: Department of Ophthalmology, County Council Hospital, Ryhov, Jönköping, Sweden. METHODS: Twenty-four eyes in 23 patients with keratoconus had photorefractive keratectomy (PRK) to reduce the steepness of the cone. A VISX Twenty-Twenty B laser (193 nm) was used for the treatments. Spherical ablations, cylindrical ablations, or both were used. Patients were followed for a mean of 22 months (range 6 to 46 months). RESULTS: Fourteen patients (58%) had improved visual acuity. Eleven (46%) could manage with only spectacles, and three (13%) were fitted with contact lenses to get adequate visual function. All treated corneas healed, and no acceleration of the keratoconus was seen. CONCLUSION: No increased risk was associated with treating primary keratoconus with excimer laser PRK. We found that excimer laser surgery can improve vision and the ability to wear contact lenses, and it did not interfere with subsequent corneal transplantation surgery.

Cornea↗
PubMed1998

Simultaneous confocal lifetime imaging of multiple fluorophores using the intensity-modulated multiple-wavelength scanning (IMS) technique.

We demonstrate the simultaneous recording of confocal lifetime images of multiple fluorophores. The confocal microscope used in the study combines intensity-modulated laser illumination, lock-in detection and spectral separation of the fluorescent light. A theoretical investigation is presented that describes how the signal-to-noise ratio (SNR) depends on various factors such as modulation frequency, degree of modulation and number of detected photons. Theory predicts that, compared with ordinary intensity images, lifetime images will have a SNR that is, at best, approximately four times lower. Experimental results are presented that confirm this prediction.

Journal Article↗
PubMed1997

High-dose chemotherapy with autologous stem cell support for the treatment of advanced ovarian cancer--initial experience in Uppsala and Turku.

BACKGROUND: With current standard-dose chemotherapy ovarian cancer is a chemosensitive but not chemocurable disease in the majority of cases. The widely used first-line chemotherapy including a platinum analogue combined with cyclophosphamide results in response rates of 60-80%. However, only 10-20% of patients with advanced disease are alive 5 years after the diagnosis. The efficacy of high-dose chemotherapy supported by autologous stem cell transplantation (ASCT) is currently under intensive investigation. METHODS: We report here our initial experiences of the use of high-dose chemotherapy supported by ASCT for patients with high-risk ovarian cancer. Two patients were treated at Uppsala University Hospital in 1992 and four patients at Turku University Central Hospital in 1994. RESULTS: The first four patients treated either after heavy previous chemotherapy or recurrent disease relapsed within 5-10 months. Two patients received high-dose therapy as part of first-line treatment. One of them had a relapse 18 months after therapy, the other one has been disease free for 28 months. No toxic deaths occurred, but the patients had neutropenic febrile episodes and moderate to severe gastrointestinal toxicity. CONCLUSIONS: Coordinated efforts in Nordic countries are indicated to evaluate the usefulness of high-dose therapy supported by ASCT in the treatment of advanced ovarian cancer.

Adult↗
PubMed1996

[Transport and localization of troxerutin in the venous wall].

The vein wall is nourished by diffusion of blood nutrients from the lumen and from the vasa vasorum. It is likely that drugs take the same ways to reach and diffuse through the vessel wall. Thus the uptake of a drug with affinity for the vein wall should give information on its transport to the tissue. This study aimed to explore troxerutin uptake by the long saphenous vein. Troxerutin is a naturally fluorescent flavonoid which has been known to improve subjective signs of patients with chronic venous insufficiency. Nine patients undergoing surgical treatment of varicosis were enrolled in the study. They received for the last 4 days before surgery either 3,500 mg of troxerutin (n = 4) or 1,000 mg twice daily (n = 2). Three patients as controls did not receive any drug. Two samples from thigh and calf long saphenous vein were harvested in each patients and investigated with a confocal laser scanning microscope developed by our institute measuring the fluorescence emitted by troxerutin after excitation by a 458 nm wavelength laser-beam. The intensity of the overall fluorescence was significantly higher in the treated groups (p < 0,001) and slightly higher in the patient who received 3,500 mg of troxerutin than with the lower dosage. The outer wall region provided the highest fluorescence in the treated group while a significant difference was observed in the fluorescence of the medial region between treated and control group. These results showed a marked affinity of troxerutin for the venous wall. The highest uptake in the outer wall region is likely to result from transport through vasa vasorum, owing to the rheologic properties of the drug. The significant medial fluorescence may account for the venous tone improvement with the drug.

Endothelium, Vascular↗
PubMed1995

Improved fluorophore separation with IMS confocal microscopy.

Some of the most extensively used fluorophores for multicolour fluorescence labelling of tissue, such as Lucifer yellow, fluorescein, Cyanine-3.18 and rhodamine, all have the disadvantage of being difficult to separate spectrally due to their broad and overlapping emission spectra. With the intensity-modulated multiple-beam scanning (IMS) technique, cross-talk between labels can be reduced to a fraction of that seen using conventional excitation and detection technique. Thus, cross-talk to the 'red' channel decreased with about one order of magnitude for the 'green' fluorophores Lucifer yellow and FITC. For the tested 'red' fluorophores, the amount of cross-talk to the green channel was reduced by 60-90%.

Animals↗
PubMed1995

Fibrinogenemia Tampere--a dysfibrinogenemia with defective gelation and thromboembolic disease.

Fibrinogen Tampere was found in a woman with severe thromboembolic disease. The thrombin induced clotting time of her plasma and purified fibrinogen was slightly prolonged. The activation of fibrinogen Tampere appeared to be normal but subsequent gelation was defective. We studied fibrin gels formed at different ionic strengths and at different fibrinogen and calcium concentrations by liquid permeation, turbidity, and 3D laser microscopy. Crosslinking was studied by SDS-gel electrophoresis. The gels formed from fibrinogen Tampere were at ionic strength above 0.2 much tighter and had lower fiber mass-length ratios than normal gels as judged by permeability and turbidity data. At ionic strength 0.15 and at different calcium concentrations analysis by permeability showed the same results for fibrinogen Tampere as for normal gels. Analysis by turbidity at ionic strength 0.15 suggested swelling of the fibers at low calcium concentrations. 3D microscopy revealed perturbed clot architecture under all conditions. In fibrin gels from fibrinogen Tampere, the gamma-chain crosslinking was normal but the crosslinking of alpha-chains was delayed at ionic strength 0.2 and also at lower ionic strengths on lowering the calcium concentration. The abnormal gelation may be due to a mutation in the fibrinogen molecule. Tendency to form tight fibrin gels and/or insufficient crosslinked fibrin matrix may be pathogenetic in this thrombotic disease.

Adult↗
PubMed1995

A confocal scanning laser microscope for quantitative ratiometric 3D measurements of [Ca2+] and Ca2+ diffusions in living cells stained with Fura-2.

A confocal scanning laser microscope (CSLM) for observation and quantitative ratiometric measurements of the intracellular dynamics of Ca2+ ions in living neurons has been developed. The instrument consists of a UV-enhanced CSLM, an optical arrangement providing simultaneous excitation at two wavelengths, an electronic arrangement for processing the simultaneous fluorescence response, and software for computing the absolute Ca2+ concentrations, ([Ca2+]). The instrument can be used for any excitation ratiometric measurements, provided that the dye substance used is excitable by wavelengths between 334 nm and 750 nm (such as, e.g. Fura-2). The spatial resolution of the CSLM, as well as a temporal resolution of 20 ms per line (maximum sampling rate) for dynamic measurements are provided by the instrument. Using Fura-2 in calibrated Ca2+ buffer solutions, the instrument measures [Ca2+] between 0 and 1.35 mumol.l-1 with an error of less than 1%. The capability of the instrument to measure absolute [Ca2+] was verified by recording fluorescence images of test solutions with well defined [Ca2+] values (Molecular Probes, Eugene, Ore., USA, C-3009 calibration solutions). In order to verify the dynamic capability of the instrument in real biological specimens, fluorescence changes of Fura-2 that were due to an intracellular flux of Ca2+ ions, and to an increase of [Ca2+]i (the intracellular Ca2+ concentration) have been recorded in Fura-2-loaded cultured cells of the line TE 671.

Calcium↗