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  • 201.
    Lerm, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Holm, Åsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Seiron, Å.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, E.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Descoteaux, A.
    INRS- Institut Armand-Frappier, Université du Québec, Laval, Québec, Canada.
    Rasmusson, Birgitta
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Rac1 and Cdc42 are involved in the periphagosomal F-actin accumulation and inhibition of phagosomal maturation caused by Leishmania donovani lipophosphoglycanManuscript (preprint) (Other academic)
    Abstract [en]

    The intracellular parasite Leishmania donovani survives inside macrophage phagosomes by inhibiting phagosornal maturation. Its main surface glycoconjugate, lipophosphoglycan (LPG), is crucial for survival and essential for the build-up of a coat of F-actin surrounding the phagosome. Previous studies have shown that inhibition of PKCα by LPG is partly responsible for the elevated levels of F-actin around the phagosome (1, 2). This study shows that simultaneous inhibition of Cdc42 and Rac1, members of the Rho family of small GTPases, prevented the accumulation of F-actin around L. donovani containing phagosomes in murine macrophages. Moreover, an LPG-defective L. donovani mutant normally not capable of accumulating F-actin around it's phagosome, displayed elevated amounts of periphagosomal F-actin in cells pre-treated with permanently active forms of Cdc42 and Rac. The lysosomal marker LAMP1 did not translocate normally to phagosomes in these cells, indicating defective phagosomal maturation. We conclude that Cdc42 and Rac are activated by L. donovani in an LPG-dependent manner, and that this activation contributes to the accumulation of periphagosomal F-actin around L. donovani phagosomes. Our results also indicate a direct link between the build-up of periphagosomal F-actinand inhibition of phagosomal mahuation.

  • 202.
    Li, Zhao-Qi
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Mechanisms of secretagogue action in isolated parietal cells1995Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The mechanisms of secretagogue action and various intracellular events in isolated pig and rat gastric parietal cells were investigated.

    The response to gastrin in aminopyrine accumulation, an index of the acid produced and n·apped by the cells, was ilifferent in pig and rat parietal cells. In pig, gastrin alone stimulated (unaffected by H2 antagonist ranitidine) and potentiated the action of histamine, IBMX, DBcAMP and Sp-cAMP[S]. In rat cells, gastrin alone was ineffective but potentiated the actions of histamine, DBcAMP and Sp-cAMP[S]. The stimulation of aminopyline accumulation by the acetylcholine analogue carbachol, or by gastrin (in pig), was dosedependently inhibited by the protein kinase A inhibitor Rp-cAMP[S]. In rat cells, histamineandDBcAMP-stimulated aminopyrine accumulations were dose-dependently inhibited by the intracellular Ca2+ chelator BAPT A.

    The basal intracellular cAMP content in pig palietal cells was 3.5-fold higher than that in rat parietal cells. Although IBMX slightly increased cAMP content, it was not enough to increase aminopyrine accumulation in rat. Histamine combined with IBMX increased the cAMP content by 8- to 38-fold. The aminopyrine accumulation, however, was not stimulated further than that observed with histamine-stimulation alone.

    Gasn·in increased cytosolic free Ca2+ in both pig and rat palietal cell. Basal Ca2+ was reduced by the intracellular Ca2+ chelator BAPTA, and both gasn·in- and carbachol-induced increases in cytosolic free Ca2+ were abolished by the inclusion of BAPT A.

    Gastrin was as efficient as histamine in inducing the formation of vacuolar/canalicular spaces in the parietal cells, i.e., inducing a secretion-associated morphology. Ranitidine abolished histamine- but not gasn·in-induced morphological n·ansformation. In the presence of BAPTA, the morphological transformations induced by either gastrin, the cAMP analogues DBcAMP or Sp-cAMP[S] were completely abolished.

    It is concluded that: I) gastrin has a direct action on the parietal cells; 2) species difference of gastrin action seems to be related to different basal cAMP contents; 3) Ca2+. dependent morphological transformation is essential for aminopyrine accumulation; and 4) a threshold level of either Ca2+ or cAMP seems to be required for the stimulation by the other second messenger.

  • 203.
    Lillesaar, Christina
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Molecular factors influencing nerve growth: studies on the developing rodent trigeminal ganglion and tooth pulp2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The adult tooth pulp has a rich protective sensory innervation from the trigeminal ganglion (TG). The developmental establishment of this exclusively nociceptive innervation is not fully understood today. However, an increasing amount of data indicates that the tooth pulp cells produce molecular factors, which attract relevant nerve fibres from adjacent major nerve trunks. Presently, a wide variety of agents, which influence the growth of nerve fibres in various contexts, are known. Molecular factors, which might influence the innervation process of the developing tooth, are the focus of this thesis.

    Based on gene-expression studies it has been suggested that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) may be involved in the formation of a pulpal innervation. However, these studies did not examine how pulpal tissue/pulpal cells and TGs/TG neurones actually interact in vitro. In the first of the four studies included in this thesis we show that pulpal explants from neonatal rat pups elicit neurite growth from eo-cultured TG explants. It was also fotmd that application of exogenous NGF or GDNF, but not BDNF, to single TGs in vitro gives a rich neurite outgrowth. However, treatment of TG/pulpal co-cultures with antibodies against these neurotrophic factors does not fully block the neuritogenic effect of the pulpal explant. This seems to suggest that other, as yet tm.known factors are also involved. The second paper supplements the first one by illustrating the pulpal/TG interaction at the level of single cells. The results show that single TG neurones emit relatively linear neurites up to a single pulpal cell or a group of such cells. Upon reaching the pulpal cell/cells the neurite changes behaviour and forms a network of thin beaded branches in direct relation to the cell/cells. In co-cultures of TG neurones and a mixture of pulpal cells and 3T3 fibroblasts the neurones form neuritic networks exclusively in relation to the former. Finally, RT/PCR analysis revealed that both pulpal tissue and cultured pulpal cells express the genes for all known mammalian neurotrophins and GDNF-family members. The third paper compares the effects of cultured pulpal cells, and transfected 3T3 fibroblasts overexpressing selected neurotrophic factors on single TG neurones in terms of survival and neurite growth. It was found that the different 3T3 fibroblasts affect the neurones in different ways, and that none of them fully mimic the influences of wild-type pulpal cells. Also, the different 3T3 fibroblasts induce dissimilar neurite branching patterns. The pattern associated with NGF-overexpressing 3T3 cells is most similar to the one seen in co-cultures with TG neurones and pulpal cell.

    Onset of nerve growth to the tooth pulp occurs comparatively late in development, although tooth pulp cells produce neurite growth stimulating molecular factors already during embryonic development, and in spite of the fact that nerve fibres from neurones expressing the appropriate receptor genes are located in the vicinity. These observations suggested the hypothesis that axon growth to prospective tooth pulps is transiently actively blocked during embryonic and early neonatal development. But, when amelogenesis and dentinogenesis have commenced nerve fibres enter the tooth pulp. This indicates that the hypothetical blockade decreases after birth, allowing an innervation of the tooth pulp to become established. In order to test this hypothesis, tooth-related tissue and TG explants from mice at various developmental stages were eo-cultured. It was found that mesenchymal tissue collected from early developing jaw or tooth primordia repel TG neurites, and that corresponding tissue from teeth in a more advanced stage of development attract TG neurites. One group of molecules with neurite growth-inhibiting properties are the semaphorins. Hence, we examined the presence of mRNAs for selected semaphorins in mesenchymal tissue collected from the jaw or from tooth pulps at different developmental stages. The results show that some of the selected semaphorins are expressed in a temporal pattern that matches the observed neuriteinhibiting effects seen in vitro and that conforms with the idea that there is an early pulpalderived blockage of nerve fibre growth in situ. Also, it was found that nerve fibres in the vicinity of the early developing tooth are immunoreactive for a relevant semaphorin receptor, neuropilin-1. After birth, when amelogenesis and dentinogenesis have commenced, nerve fibres in the tooth pulp are immunonegative for that receptor.

    To conclude, our results show that pulpal tissue/pulpal cells can influence nerve fibres from TG explants/TG neurones in vitro. Also, we provide evidence that neurotrophins, GDNF-family members and semaphorins may contribute to this influence.

  • 204.
    Lillesaar, Christina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Arenas, E.
    Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics Karolinska Institutet, Stockholm, Sweden.
    Hildebrand, Claes
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fried, K.
    Center for Oral Biology, Novum, Karolinska Institutet, Huddinge, Sweden.
    Responses of rat trigeminal neurones to dental pulp cells or fibroblasts overexpressing neurotrophic factors in vitro2003In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 119, no 2, p. 443-451Article in journal (Refereed)
    Abstract [en]

    The adult dental pulp is innervated by sensory trigeminal axons and efferent sympathetic axons. Rat trigeminal ganglia extend neurites when co-cultivated in vitro with pulpal tissue explants, suggesting that pulpal cells secrete soluble molecules that stimulate the growth of trigeminal ganglion axons. In addition, cultured pulpal cells produce mRNAs for neurotrophins and glial cell line-derived neurotrophic factor-family members. These data suggest that neurotrophic factors are involved in the formation of a pulpal innervation. Here, we examine how pulpal cells and 3T3 fibroblasts overexpressing certain neurotrophic factors (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, glial cell line-derived neurotrophic factor or neurturin) influence survival and growth of single trigeminal ganglion neurones in vitro in quantitative terms. The results show that most of the neurotrophic factor-overexpressing fibroblasts induce similar neuronal soma diameters, but higher survival rates and neurite lengths compared with pulpal cells. With respect to neurite growth pattern, trigeminal ganglion neurones co-cultured with fibroblasts overexpressing nerve growth factor develop a geometry that is most similar to that seen in co-cultures with pulpal cells. We conclude that none of the fibroblasts overexpressing neurotrophic factors can fully mimic the effects of pulpal cells on trigeminal ganglion neurones, and that nerve growth factor promotes a neurite growth pattern most similar to the picture seen in co-cultures with pulpal cells.

  • 205.
    Lillesaar, Christina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Eriksson, C.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fried, KJ
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Rat tooth pulp cells elicit neurite growth from trigeminal neurones and express mRNAs for neurotrophic factors in vitro2001In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 308, no 3, p. 161-164Article in journal (Refereed)
    Abstract [en]

    Molecular factors control the developmental ingrowth of axons to the tooth pulp. Here we examine the ability of pulpal cells to induce neurite outgrowth from neonatal rat trigeminal neurones (TGNs) in vitro. We found that TGNs emitted neurites and formed networks of branches in relation to pulpal cells. Neurones co-cultured with a mixture of pulpal cells and 3T3 fibroblasts formed networks exclusively in relation to the pulpal cells. Cultivated pulpal cells and pulpal tissue produced mRNAs for all neurotrophins and members of the glial cell line-derived neurotrophic factor family. Hence, rat pulpal cells have neuritogenic effects on single TGNs in vitro, that may be associated with secretion of neurotrophic factors.

  • 206.
    Lillesaar, Christina
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Eriksson, Cecilia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Johansson, Carina S.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fried, Kaj
    Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
    Hildebrand, Claes
    Linköping University, Department of Clinical and Experimental Medicine, Cell Biology. Linköping University, Faculty of Health Sciences.
    Tooth pulp tissue promotes neurite outgrowth from rat trigeminal ganglia in vitro1999In: Journal of Neurocytology, ISSN 0300-4864, E-ISSN 1573-7381, Vol. 28, no 8, p. 663-670Article in journal (Refereed)
    Abstract [en]

    The mammalian tooth pulp becomes innervated by nociceptive and sympathetic axons relatively late during development, when part of the root has formed. In the adult, regenerating axons from an injured tooth nerve or sprouting axons from uninjured nerves in the vicinity rapidly reinnervate denervated tooth pulps. These observations indicate that tooth pulp tissue can use molecular factors to attract pulpal axons from local nerve trunks. The present study examines the hypothesis that these factors include nerve growth factor (NGF), brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF). Explants of trigeminal ganglia from neonatal rat pups showed a distinct neurite outgrowth when co-cultured with pulpal explants collected from molar teeth of 12-day old pups, or after application of a pulpal extract. Control cultures, containing single ganglionic explants, or explants co-cultured with heat-treated pulpal tissue, exhibited a sparse neurite outgrowth. Exogenous NGF and/or GDNF, but not exogenous BDNF, stimulated neurite outgrowth from ganglionic explants. Unexpectedly, application of antibodies against NGF, BDNF and/or GDNF to co-cultures of ganglionic and pulpal explants did not inhibit neuritogenesis. Control experiments showed that IgG molecules readily penetrate the gel used for culture and that even very high concentrations of NGF and GDNF antibodies in combination failed to block neurite growth. On the basis of these data we suggest that other as yet unknown neurite-promoting factors might be present and active in TG/pulpal co-cultures.

  • 207.
    Lillesaar, Christina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hildebrand, Claes
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Denervation does not affect the growth of rat vibrissae1999In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 261, no 1-2, p. 69-72Article in journal (Refereed)
    Abstract [en]

    This study examines the hypothesis that neural factors influence the growth of rat vibrissae. We divided the vibrissae in rows α-δ, 1 and 2 and examined their regrowth during the first complete growth period in normal and nerve-lesioned rats. The lesions used were denervation through neonatal capsaicin treatment, surgical sympathecomy in adult rats, neurectomy of the mandibular and buccal branches of the facial nerve in adult rats or division of the infraorbital nerve in adult rats. Normal vibrissae developed a length of 51.1 mm and a diameter of 178 μm (row α-δ), 44.1 mm and 181 μm (row 1) and 33.2 mm and 165 μm (row 2). In all experimental groups the examined vibrissae developed a normal final length and proximal diameter. This indicates that local nerves do not influence vibrissal growth to any major extent.

  • 208. Lindahl, Marika
    et al.
    Spetea, Cornelia
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Hundal, Torill
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Oppenheim, Amos
    Adam, Zach
    Andersson, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein2000In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 12, no 3, p. 419-431Article in journal (Refereed)
    Abstract [en]

    The photosystem II reaction center D1 protein is known to turn over frequently. This protein is prone to irreversible damage caused by reactive oxygen species that are formed in the light, the damaged, nonfunctional D1 protein is degraded and replaced by a new copy. However, the proteases responsible for D1 protein degradation remain unknown. In this study, we investigate the possible role of the FtsH protease, an ATP-dependent zinc metalloprotease, during this process. The primary light-induced cleavage product of the D1 protein, a 23-kD fragment, was found to be degraded in isolated thylakoids in the dark during a process dependent on ATP hydrolysis and divalent metal ions, suggesting the involvement of FtsH. Purified FtsH degraded the 23-kD D1 fragment present in isolated photosystem II core complexes, as well as that in thylakoid membranes depleted of endogenous FtsH. In this study, we definitively identify the chloroplast protease acting on the D1 protein during its light-induced turnover. Unlike previously identified membrane-bound substrates for FtsH in bacteria and mitochondria, the 23-kD D1 fragment represents a novel class of FtsH substrate - functionally assembled proteins that have undergone irreversible photooxidative damage and cleavage.

  • 209. Lindberg, Jan
    et al.
    Strålfors, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Konradsson, Peter
    Synthesis of inositol phosphoglycans containing thiol-terminated spacers for efficient coupling to maleimide functionalized solid phases or proteins2002In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 58, no 21, p. 4245-4254Article in journal (Refereed)
    Abstract [en]

    The synthesis of inositol phosphoglycans (IPGs), analogous to second messengers of insulin, to provide a small targeted library of compounds is described. These derivatives contain the glucosamine(a1-6)myo-inositol 1,2-cyclic phosphate motif. A thiol-terminated spacer was introduced, for their immobilization, by a radical elongation of an allyl ether with benzyl mercaptane. ⌐ 2002 Elsevier Science Ltd. All rights reserved.

  • 210. Lindberg, Mikael
    et al.
    Tibell, Lena
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Oliveberg, Mikael
    Commmon denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: Decreased stability of the apo state2002In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, p. 16607-16612Article in journal (Refereed)
  • 211.
    Lindqvist Appell, Malin
    et al.
    Linköping University, Department of Medicine and Care, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Gastroenterology and Hepatology. Östergötlands Läns Landsting, MC - Medicincentrum, EMT-magtarm.
    Peterson, Curt
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medicine and Care, Clinical Pharmacology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Real-time RT-PCR methodology for quantification of thiopurine methyltransferase gene expression2003In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 59, no 3, p. 207-211Article in journal (Refereed)
    Abstract [en]

    Objective: The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells. Methods: TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6- 14C-methylmercaptopurine from 6-MP using S-adenosyl-L-( 14C-methyl)-methlonine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity. Results: Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (rs = 0.66, P = 0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population. Conclusion: We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.

  • 212.
    Lindström, Torbjörn
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Frystyk, Jan
    The Medical Research Laboratories, Clinical Institute and Medical Department M, Aarhus University Hospital, Aarhus, Denmark.
    Hedman, Christina
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Flyvbjerg, Allan
    The Medical Research Laboratories, Clinical Institute and Medical Department M, Aarhus University Hospital, Aarhus, Denmark.
    Arnqvist, Hans
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Elevated circulating adiponectin in type 1 diabetes is associated with long diabetes duration2006In: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 65, no 6, p. 776-782Article in journal (Refereed)
    Abstract [en]

    Objective  To study circulating adiponectin concentrations in relation to diabetes duration and endogenous insulin secretion in patients with type 1 diabetes.

    Patients  Patients with haemoglobin A1c (HbA1c) < 6% (reference range 3·6–5·4%) were selected for the study. Twenty-two men and 24 women [age 41·3 ± 13·8 years (mean ± SD), diabetes duration 4 months to 52 years] participated. Healthy controls (15 women and nine men, age 41·3 ± 13·0 years) were also included. Overnight fasting serum samples were analysed for adiponectin, HbA1c, C-peptide and lipoproteins.

    Results  Significant positive associations were found between adiponectin concentrations and diabetes duration in univariate and multiple regression analyses. Serum adiponectin averaged 9·7 ± 5·3 [median 8·1, interquartile range (IQR) 3·6] mg/l in patients with diabetes duration less than 10 years and 17·8 ± 10·7 (median 14·7, IQR 7·5) mg/l in patients with longer duration (P = 0·0001). Among the patients, 24 were without detectable (< 100 pmol/l) and 22 with detectable C-peptide levels (185 ± 91 pmol/l). C-peptide levels in controls averaged 492 ± 177 pmol/l. HbA1c was 5·7 ± 0·6% in patients without detectable C-peptide and 5·6 ± 0·4% in patients with detectable C-peptide (ns). Serum adiponectin was higher in patients without detectable C-peptide than in patients with detectable C-peptide [17·3 ± 11·1 vs. 10·6 ± 5·8 mg/l (P < 0·005)] and in the controls [10·1 ± 2·9 mg/l (P < 0·001 vs. patients without detectable C-peptide)].

    Conclusions  The increase in circulating adiponectin concentrations in patients with type 1 diabetes appears to be strongly associated with long diabetes duration, irrespective of the metabolic control. Among other factors, a putative role for residual β-cell function in the regulation of circulating adiponectin levels can be considered but we did not find sufficient evidence for this in the present study.

  • 213.
    Lindström, Torbjörn
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Hedman, Christina
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Arnqvist, Hans
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Use of a novel double-antibody technique to describe the pharmacokinetics of rapid-acting insulin analogs2002In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 25, no 6, p. 1049-1054Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE—To measure the contribution of bedtime intermediate-acting human insulin on the morning plasma insulin profiles after injection of the rapid-acting insulin analogs lispro and aspart in patients with type 1 diabetes.

    RESEARCH DESIGN AND METHODS—A total of 14 patients with type 1 diabetes, aged 35 ± 13 years (mean ± SD), participated in this single-blind, randomized crossover study. After taking their usual injection of human intermediate-acting insulin the night before, they were given insulin aspart or insulin lispro (10 units) before a standardized breakfast. The contribution of continuing absorption of the human insulin was measured using a monoclonal antibody not cross-reacting with insulin aspart or lispro, whereas the contribution of the analogs was estimated by subtraction after measurement of all plasma free insulin using an antibody cross-reacting equally with human insulin and both analogs.

    RESULTS—The correlation coefficient of the fasting free insulin concentrations measured with both insulin methods was 0.95. Fasting free insulin was 95 ± 25 pmol/l before administration of insulin aspart, when determined with enzyme-linked immunosorbent assay detecting only human insulin, and 71 ± 20 pmol/l before administration of insulin lispro (NS). Both insulin analogs gave marked peaks of free insulin concentrations, lispro at 40 ± 3 min and aspart at 55 ± 6 min after injection (P = 0.01). The later part of the profiles, from 4.5 to 5.5 h after injection, were similar and showed almost no contribution of the insulin analogs.

    CONCLUSIONS—The combination of insulin assays that detect human insulin only or both human insulin and analogs provides a new tool for studying insulin pharmacokinetics. Using this technique, we showed that 4.5 h after administration of the rapid-acting insulin analogs lispro and aspart, the free insulin levels are almost only attributable to the intermediate-acting insulin given at bedtime.

  • 214.
    Lirvall, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dynamics and gene expression of growth factor receptors in human cultured skin cells: Effects of UV radiation and calcium on EGF- and PDGF-receptors2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Growth factors transmit biological signals for the stimulation of cell growth and their stimulation may be involved in turnourigenesis. It is therefore of great importance to understand growth factor receptor reactions in response to stimuli such as calcium depletion or ultraviolet radiation, which normal human cells are invariably exposed to during their growth cycle. We have studied human skin cells i.e. fibroblasts, lceratinocytes and melanocytes and their growth factor receptor expression on the surface of cells, reactions in the plane of the cell membrane, intracellular trafficlcing, and gene expression after exposure to their ligand, serum, UVB radiation and calcium depletion. We have used Fluorescence recovery after photo bleaching (FRAP) to assess receptor characteristics in cell membranes, confocal laser scanning microscopy to visualize receptor internalization, Ratio imaging for calcium studies, Northern blot for detection of the gene for the epidermal growth factor receptor (EGF-R) and flow cytometry for cell surface receptor determination. Platelet-derived growth factor receptors (PDGF-R) and EGF-R were studied since they affect cell proliferation and viability.

    Our results showed that the addition of PDGF increased receptor mobility characteristics in normal fibroblasts, also "starvation" of cells increased their receptor mobility. We could show that changes in both intra-and extracellular free Ca2+ influence the mobility characteristics of PDGF-ß2 receptors. We have demonstrated that the three celltypes display different basal EGF-R mobility characteristics. After UVB irradiation mobility characteristics increased in all cell types but with differences in diffusion coefficients or mobile fractions. Addition of antioxidant enzymes, catalase and superoxide dismutase, prior to UVB irradiated cells abolished the UY-induced receptor mobility changes. We have shown that a single physiologic dose ofUVB radiation alters the intracellular EGF-R distribution and intracellular transport in melanocytes. It also significantly alters the melanocyte phenotype. We were able to detect a constitutive EGF-R gene expression and showed that UVB-radiation induces a time-dependent induction in EGF-R mRNA in melanocytes. Human melanocytes express EGF-R on their cell surface and UVR induces time dependent changes in the number of receptors but the number of receptors does not correlate with the level of UV-induced EGF-R gene expression.

    It is concluded that UV-radiation, growth factors and calcium, ubiquitous constituents of every day life, all having tremendous effects in vivo, also affect human cells in vitro in parameters studied that are of importance for proliferation, survival and tumourigenesis.

  • 215.
    Lirvall, Margareta
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hallbeck, Anna-Lotta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Briheim, Kristina
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    UVB radiation increases EGF-R gene expression in cultured human normal melanocytesManuscript (preprint) (Other academic)
    Abstract [en]

    Epidermal growth factor (EGF) and its receptor are thought to play important roles in the UV response of mammalian cells. Since UVB radiation has been shown to influence melanocyte growth and development, we sought to determine whether UVB would alter the expression of the gene for EGF-R. Using Northern blot, the present study exanlined the expression of EGF-R mRNA in cultured normal human melanocytes exposed to a single physiologic dose of UVB radiation. Total cellular RNA was extracted from cultured normal human melanocytes at various time-points after irradiation or sham-irradiation. Constitutive expression ofEGF-Rmfu'!A was detected in the melanocytes. After UVB irradiation with a single dose of 6o or 180 mJ/cm2, the EGF-R rnRNA decreased within 12 h to approximately 50 % of the initial value. After 24 h there was a subsequent increase and a peak at 36 h, showing a three-fold increase. After 48 hours a decline was seen, but the expression of EGF-R mRNA was still doubled as compared to control.

    Using flow cytometric analysis EGF-R on melanocytes were detected. The median EGF-R immunofluorescence was decreased after UVB-irradiation as compared to non-irradiated cells. The lowest EGF-R immunofluorescence was seen 12 hours after UVB irradiation. Witi1in 48 hours post irradiation we could not detect any increase in EGF-R that we would correlate to the increased EGFR gene expression seen at 36 hours. We conclude ti1at the steady-state levels of EGF-R mRNAis upregulated by single exposure to UVB.

  • 216.
    Lirvall, Margareta
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ljungquist-Höddelius, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    UVB radiation affects the mobility of epidermal growth factor receptors in human keratinocytes and fibroblasts1996In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 16, no 3, p. 227-238Article in journal (Refereed)
    Abstract [en]

    Growth factor receptors transmit biological signals for the stimulation of cell growth in vitro and in vivo and their autocrine stimulation may be involved in tumorigenesis. It is therefore, of great value to understand receptor reactions in response to ultraviolet (UV) light which certain normal human cells are invaribly exposed to during their growth cycle. UV irradiation has recently been shown to deplete antioxidant enzymes in human skin. The aims of the present study were a) to compare the lateral mobility of epidermal growth factor receptors (EGF-R) in cultured human keratinocytes and human foreskin fibroblasts, b) to investigate effects of ultraviolet B radiation on the mobility of EGF-R in these cells, and c) study the response of EGF-R on addition of antioxidant enzymes. The epidermal growth factor receptors were labeled with rhodaminated EGF, the lateral diffusion was determined and the fraction of mobile EGF-R assessed with the fluorescence recovery after photobleaching (FRAP). We found that human keratinocytes display a higher basal level of EGF-R mobility than human skin fibroblasts, viz, with diffusion coefficients (D ± standard error of the mean, SEM) of 4.2 ± 0.2 x 10-10 cm2/s, and 1.8 ± 0.2 x 10-10 cm2/s, respectively. UVB-irradiated fibroblasts showed an almost four-fold increase in the diffusion coefficient; D was 6.3 ± 0.3 x 10-10 cm2/s. The keratinocytes, however, displayed no significant increase in receptor diffusion after irradiation; D was 5.1 ± 0.8 x 10-10 cm2/s. In both cell types the percentage of EGF-R fluorescence recovery after photobleaching, i.e. the fraction of mobile receptors, was significantly increased after irradiation. In keratinocytes it increased from 69% before irradiation to 78% after irradiation. Analogous figures for fibroblasts were 61% and 73%. The effect of UVB on fibroblast receptors was abolished by prior addition of superoxide dismutase (SOD) and catalase (CAT). It is concluded that UVB radiation of fibroblasts and keratinocytes can affect their biophysical properties of EGF-R. The finding that addition of antioxidant enzymes prevented the UVB effect in fibroblasts may indicate the involvement of reactive oxygen metabolites.

  • 217.
    Lirvall, Margareta
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Majeed, Meytam
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Höddelius, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Rosdahl, Inger
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    UVB radiation increases EGF receptor mobility and trafficking in human melanocytesManuscript (preprint) (Other academic)
    Abstract [en]

    For the human skin, UVB-radiation (290-320nm) is a very potent injurious agent. UV radiation is absorbed in the epidermis and reaching the melanocytes leads to proliferation via activation of growth factor reccptors. This may play a key role in the clonal expansion of melanocytes and be a critical step in carcinogenesis. We show that UVB-irradiated human epidermal melanocytes display an increased mobility of epidermal growth factor receptors (EGF-R) in the plane of the cell membrane, and that UVB affects the intracellular EGF-R transport to the nucleus. Using fluorescence photobleaching technique we show a time and dose dependent increase in the diffusion coefficient and mobile fraction of EGF-R. EGF-Rdiffuse with a low rate within the cell membrane in control cells, and the mobility increases 4-fold after single physiologic doses of UVB. Three-dimensional confocal microscopy reveals that EGF-R display a strilting difference in receptor distribution and intracellular transport before and after UVB irradiation. The EGF-Rclearly eo-localize with clathrin-coated pits within the cells. These results indicate that already single physiologic doses of UVB affect growth factor receptor mobility in cell membranes and intracellular trafficlting. This may be an important early step in the ultraviolet radiation-induced signal transduction pathway leading to cell proliferation.

  • 218. Littorin, B
    et al.
    Nyström, L
    Gullberg, B
    Råstam, L
    Östman, J
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology. Östergötlands Läns Landsting, MC - Medicincentrum, EMT-endo.
    Björk, E
    Blohmé, G
    Bolinder, J
    Eriksson, JW
    Scherstén, B
    Sundkvist, G
    Increasing body mass index at diagnosis of diabetes in young adult people during 1983-1999 in the Diabetes Incidence Study in Sweden (DISS)2003In: Journal of Internal Medicine, ISSN 0954-6820, E-ISSN 1365-2796, Vol. 254, no 3, p. 251-256Article in journal (Refereed)
    Abstract [en]

    Objective. To study trends in body mass index (BMI) at diagnosis of diabetes in all young Swedish adults in the age range of 15-34 years registered in a nation-based registry. Design. The BMI was assessed at diagnosis in diabetic patients 15-34 years of age at diagnosis, for a period of 17 years (1983-1999). Islet cell antibodies (ICA) were measured during three periods (1987-1988, 1992-1993 and 1998-1999). Setting. A nationwide study (Diabetes Incidence Study in Sweden). Subjects. A total of 4727 type 1 and 1083 type 2 diabetic patients. Main outcome measures. Incidence-year specific BMI adjusted for age, gender and time of diagnosis (month). Results. Body mass index at diagnosis increased significantly both in type 1 (21.4 ▒ 3.6 to 22.5 ▒ 4.0: P < 0.0001) and in type 2 (27.4 ▒ 6.8 to 32.0 ▒ 6.0, P < 0.0001) diabetic patients, also when adjusted for age, gender and month of diagnosis. A similar significant increase in BMI was found in type 1 diabetic patients and in type 2 diabetic patients in the periods 1987-1988, 1992-1993 and 1998-1999, years when ICA were assessed and considered in the classification of diabetes. Despite this increase in BMI, there was no increase in the incidence of diabetes in young-adult people in Sweden. Conclusion. Body mass index at diagnosis of diabetes in subjects 15-34 years of age has substantially increased during 1983-1999 in Sweden when adjusted for age, gender and month of diagnosis.

  • 219. Littorin, B
    et al.
    Sundkvist, G
    Hagopian, W
    Landin-Olsson, M
    Lernmark, Å
    Östman, J
    Blohmé, G
    Bolinder, J
    Eriksson, JW
    Lithner, F
    Scherstén, B
    Wibell, L
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Islet cell and glutamic acid decarboxylase antibodies present at diagnosis of diabetes predict the need for insulin treatment.1999In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 22, p. 409-412Article in journal (Refereed)
  • 220. Littorin, Bengt
    et al.
    Sundkvist, Göran
    Nyström, Lennarth
    Carlson, Anita
    Landin-Olsson, Mona
    Östman, Jan
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology. Östergötlands Läns Landsting, MKC-2, GE: endomed.
    Björk, Elisabeth
    Blohmé, Göran
    Bolinder, Jan
    Eriksson, Jan W
    Scherstén, Bengt
    Wibell, Lars
    Family characteristics and life events before the onset of autoimmune type 1 diabetes in young adults: A nationwide study2001In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 24, no 6, p. 1033-1037Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE - To elucidate whether family characteristics and stressful life events were associated with onset of autoimmune type 1 diabetes in young adults. RESEARCH DESIGN AND METHODS - This investigation was based on a nationwide study (Diabetes Incidence Study in Sweden) of newly diagnosed patients aged 15-34 years. Patients clinically classified as type 1 diabetic with antibodies to islet cells and/or to GAD65 were compared with age- and sex-matched control subjects via questionnaire. The questionnaire covered diabetes heredity, social environment, educational level, and life events experienced during the 12 months before diagnosis. RESULTS - The rate of response was 82% for the diabetic patients and 65% for the control subjects. Questionnaires from 349 diabetic patients and 979 control subjects were considered. Diabetes in relatives was more frequent in the patients (odds ratio [OR] 2.6) who were born in Sweden and whose mothers were of Swedish origin. No major stress factors were detected in the diabetic patients, however, in comparison with the control subjects, the diabetic patients had experienced fewer conflicts with their parents and had less often broken contacts with friends. CONCLUSIONS - Young adults with recent-onset type 1 diabetes were more exposed to heredity for diabetes, but no major prediabetic stress factors were detected. Our study does not directly support the concept that psychosocial stressful life events are involved in the development of autoimmune type 1 diabetes in young adults.

  • 221.
    Ljungquist-Höddelius, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Biophysical studies of cell membrane receptors for the growth factors PDGP and EGF: Aspects on lateral and aggregation1994Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    When extracellular growth factors bind to their specific cell surface receptors, they elicit a cascade of biochemical events, including tyrosine kinase-mediated phosphorylation and changes in intracellular free ca2+, leading to cell differentiation and proliferation. However, direct effects of ligand-binding on the receptors are poorly understood. This thesis is aimed at investigating the dynamics of growth factor receptors in human fibroblasts after ligand binding, viz. the platelet-derived growth factor (PDGF), and after addition of serum. The effect of ultraviolet B (UV-B) irradiation on the lateral mobility of epidermal growth factor (EGF) receptors was assessed in fibroblasts and keratinocytes.

    Two techniques were mainly employed to study receptor dynamics. Lateral mobility of receptors was measured with fluorescence recovery after photobleaching (FRAP), and receptor aggregation was analysed with a novel technique, image correlation spectroscopy (ICS), based on confocal laser scanning microscopy.

    The results, obtained with FRAP for the lateral mobility of the WGA-labelled glycoconjugate and PDGF receptors showed, that: i) the diffusion rate (D) and mobile fraction (R) of the PDGF-receptors depend on the growth conditions, viz. absence or presence of serum in the growthmedium, and stimulation with PDGF or serum. In general, serum increased both D and R in serum-starved fibroblasts, whereas PDGF alone did not change the mobility to any appreciable extent; ii) inhibition of tyrosine kinase using an inhibitor (Erbstatin analog) gave only a slightalteration of the lateral mobility achieved with serum or PDGF addition; iii) UV-B irradiation, noxious to skin cells, caused higher diffusion rate and mobile fraction of EGF-receptors, but in combination with the antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), the effects on D and R were counteracted; i v) modulation of ca2+ extraand/ or intracellularly, strongly affected the lateral diffusion of PDGFreceptors, as well as the response to PDGF and serum. In cells depleted intracellulary of ca2+ with a calcium chelating agent, D was reduced about 2-fold compared to control cells; after addition of serum or PDGF D increased again. When extracellular ca2+ was chelated with EGTA, R increased. In cells intra- and extracellularly depleted of ca2+, D wasslightly lower and R higher compared to the controls. In this case PDGF had no effect , whereas serum increased D and R dramatically.

    In the aggregation studies, using the novel ICS method for quantifying the aggregation state of the receptors, it was found that the surface density of the PDGF receptor clusters decreased when the temperature was raised from 4 oc to 37°C, viz. the density of the clusters was about 3-4 fold lower at 37°C than at 4°C. Tyrosine kinase inhibition with Erbstatin analog seemed to induce receptor dispersion, since the cluster density was several fold higher than without Erbstatin analog. PDGF did not induce further clustering at 37°C. PDGF partly abolished the dispersion effect of Erbstatin analog, in agreement with the involvement of tyrosine phosphorylation in PDGF stimulation of cells.

  • 222.
    Ljungquist-Höddelius, Pia
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lirvall, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Magnusson, Karl-Eric
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lateral diffusion of PDGF β-receptors in human fibroblasts1991In: Bioscience Reports, ISSN 0144-8463, E-ISSN 1573-4935, Vol. 11, no 1, p. 43-52Article in journal (Refereed)
    Abstract [en]

    When platelet-derived growth factor (PDGF) binds to its receptors a number of biochemical reactions are elicited in the cell. Several models have been presented for the effects of ligand-induced receptor conformation and aggregation on signal transduction but little is known about the direct effects on receptor diffusion. This study concerns the lateral mobility of PDGF receptors in fibroblasts. It was assessed with fluorescence recovery after photobleaching (FRAP), using rhodaminated receptor antibodies or Fab-fragments of the antibody as ligands. The aims of the investigation were: (a) to compare the lateral mobility of membrane receptors of human fibroblasts labelled with either antibodies against the PDGF receptor or Fab-fragments of the same antibodies, and (b) to study the effects of serum of PDGF on the mobility of the receptors. Human foreskin fibroblasts (AG 1523) were grown on coverslips either under standard or under serum-free conditions yielding 'normal' and 'starved' cells, respectively. Two parameters of the diffusion were evaluated; the diffusion coefficient (D) and the mobile fraction (R) of the receptors. We found that normal fibroblasts had a smaller diffusion coefficient and a lower mobile fraction compared to starved cells using antibodies for receptor labelling. The addition of PDGF, just before the measurement, increased the D and R for normal cells, while starved cells, showing higher initial values, displayed slightly reduced values of D and R. After the addition of serum, D increased and R remained low for normal cells, whereas for starved cells both D and R increased to upper limits of 11.0 x 10-10 cm2s-1 and >90% respectively. In general, the D and R values, both in normal and starved cells, were higher for cells labelled with Fab-fragments than for antibody-labelled cells. The results are discussed in relation to the natural complexity of the receptor. and how PDGF, serum, antibodies and Fab-fragments might interfere with receptor structure, aggregation state and membrane diffusion characteristics.

  • 223.
    Loborg, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    A novel method using DNA binding fluorochromes as indirect probes to study linker histone-chromatin interactions in situ 2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Over the years, a variety of methods have been used to study chromatin structure. These techniques have provided valuable information about nuclear framework, chromatin organisation, and the proteins that are involved in the formation and maintenance of chromatin. However, neither ultrastructural nor biochemical methods are suitable for investigating the dynamics of chromatin structure in structurally preserved nuclei. DNA binding fluorochromes can be used as indirect probes of chromatin structure, because their accessibility to DNA is detennined by the chromatin structure, which enables cytochemical studies of relatively undamaged cells. The aim of the work presented in this thesis was to develop a cytochemical method using the fluorocbromes 7-aminoactinomycin D (7-AAMD) and 4', 6-diamidino-2- phenylindole (DAPI) to study protein-DNA interactions in essentially intact cell nuclei.

    Both fluorochromes proved to be useful as probes of chromatin structure, particularly to study Iinker histone-chromatin interactions. Suitable protocols for permeabilisation, salt extraction of linker histones, fixation, and staining were established. Permeabilisation with digitonin followed by linker histone extraction at various NaCl concentrations and subsequent fixation in paraformaldehyde was found to enable analysis of linker histone affinity for chromatin in situ. Although the cells became fragile during the salt extraction, this protocol provided individual cells that could be measured by image cytofluorometry.

    In such analyses, DAPI had several advantages over 7-AAMD. At a concentration of 50 nM DAPI, all of the high-affinity binding sites were occupied, with only minor contributions from binding sites with lower affinity. This approach was used to study linker histone affinity in different types of cells, in cells in different stages of the cell cycle, and during cell differentiation in vivo. Our results indicate differences between the investigated cell types in regard to average Iinker histone affinity. It is generally assumed that increased linker histone affinity is associated with chromatin condensation, but that is not supported by our findings. Human fibroblasts showed substantially lower linker histone affinity for highly condensed metaphase chromatin than for interphase chromatin. Moreover, we found a tendency toward decreasing linker histone affinities in amphibian erytbroblasts that were developing into tenninally differentiated erythrocytes. However, as expected, mature avian erythrocytes showed strongly bound linker histones, probably due to their high arginine content. In conclusion, our results suggest that linker histone affinity is not directly involved in chromatin condensation.

  • 224.
    Loborg, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lindén, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lönn, Anita
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Skoglund, Per
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Rundquist, Ingemar
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    High Affinity Binding of 7-Aminoactinomycin D and 4' ,6-Diamidino-2-Phenylindole to Human Neutrophilic Granulocytes and Lymphocytes1995In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 20, no 4, p. 296-306Article in journal (Refereed)
    Abstract [en]

    The binding behavior of the DNA binding dyes 7-aminoactinomycin D (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) to human neutrophilic granulocytes and lymphocytes was studied by image cytofluorometry. Peripheral blood leukocytes were prefixed in paraformaldehyde (PFA) and attached to cover glasses. Different fixation, permeabilization, and acid extraction methods were applied before the cells were stained to equilibrium using varying concentrations of 7-AAMD or DAPI. The apparent association constant and number of high affinity dye binding sites were estimated for the different cell types, dyes, and treatments. Acid extracted cells, supposedly containing nucleosome-free DNA, were chosen to represent maximal dye binding. Only about 10% of the 7-AAMD binding sites remained in the unextracted PFA-fixed cells, and the apparent dye affinity was also reduced. We found no major difference in high affinity binding between the cell types, but granulocytes showed more fluorescence from less specifically bound 7-AAMD compared to lymphocytes. DAPI had a much higher affinity than 7-AAMD, independent of the preparation method. It showed a cooperative binding behavior with an apparent saturation of the high affinity binding sites at a dye concentration of about 50 nM. We conclude that both dyes may be useful as probes for chromatin structure in intact cells and that our new technique may contribute to such studies since it allows determination of dye affinities and numbers of high affinity binding sites in situ.

  • 225.
    Loborg, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Rundquist, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Linker histones are strongly bound to chromatin in mature avian erythrocytes and loosely bound to chromatin in mature amphibian erythrocytesManuscript (preprint) (Other academic)
    Abstract [en]

    Linker histones are known to participate in the formation and maintenance of higher order chromatin strnctnres and they are also involved in transcription control. Mature nucleated erythrocytes provide an experimental model to study linker histone binding in cells with highly condensed chromatin and extremely low transcriptional activity. The aim of this study is to investigate linker histone binding in avian and amphibian erythrocytes !mown to contain different subsets of linlcer histones. We used DAPI as an indirect cytochemical probe for the analysis of linker histone affinity for chromatin in situ. We found that linlcer histones were strongly associated to chromatin iu matnre hen erythrocytes which are known to accumulate considerable amounts of H5. Linlcer histones in matnre frog erythrocytes, known to accumulate HIo, showed significantly lower affmity for chromatin. We conclude that linlcer histone affinity is not a key factor for chromatin condensation.

  • 226.
    Loborg, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Rundquist, Ingemar
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Affinity of linker histones for chromatin in situ analyzed using DAPI as a cytochemical probe2000In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 40, no 1, p. 1-9Article in journal (Refereed)
    Abstract [en]

    Background: It is generally assumed that linker histones contribute to condensation of chromatin and to the repression of genetic activity by binding to chromatin with increased affinity. The aim of this study is to investigate a possible correlation between linker histone binding to chromatin in situ and chromatin condensation using a cytochemical approach that largely preserves nuclear structure.

    Methods: Ionically bound H1 was extracted with increasing concentrations of sodium chloride. Thereafter, the cells were fixed in formaldehyde and stained with 50 nM 4',6-diamidino-2- phenylindole (DAPI) and fluorescence intensity was measured by image cytofluorometry.

    Results: The association between linker histones and chromatin was stronger in cultured human fibroblasts and rat smooth muscle cells than in both human T lymphocytes and granulocytes, and in particular, frog erythrocytes, which exhibited highly condensed chromatin. Our data indicate a lower affinity of linker histones for metaphase chromosomes than for interphase chromatin, and we observed a reduction in linker histone affinity during terminal differentiation of frog erythrocytes in vivo.

    Conclusions: Taken together, our findings imply that the affinity of linker histones for chromatin in situ is unrelated or inversely related to chromatin condensation.

  • 227.
    Loborg, Helena
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Rundquist, Ingemar
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    DNA Binding Fluorochromes as Probes for Histone HI-Chromatin Interactions In Situ1997In: Cytometry, ISSN 0196-4763, E-ISSN 1097-0320, Vol. 28, no 3, p. 212-219Article in journal (Refereed)
    Abstract [en]

    We have investigated using the DNA binding fluorochromes 7-aminoactinomycin (7-AAMD) and 4′,6-diamidino-2-phenylindole (DAPI) as cytochemical probes for linker histone (H1)–chromatin interactions in situ. Human lymphocytes, permeabilized with digitonin, were exposed to increasing concentrations of sodium chloride to remove ionically bound H1 from the nuclei. The cells were stained to equilibrium with 1 μM 7-AAMD or 50 nM DAPI. Lymphocytes stained with 7-AAMD showed a gradual increase from 11% to 36% of HCl treated cell fluorescence intensity when the salt concentration was increased from 0.15 to 0.7 M. The corresponding increase for DAPI was 53–68%. The 7-AAMD obviously showed higher sensitivity for H1–chromatin interactions that DAPI but had disadvantages such as high background fluorescence and an affinity that was dependent on the preparation procedure. DAPI had negligible background fluorescence, and its fluorescence intensity resembles the number of available high-affinity dye-binding sites when used at 50 nM. We conclude that both fluorochromes can be used as probes for H1–chromatin interactions in situ and that our method has a potential to provide new information on such interactions.

  • 228.
    Loinder, Kristina
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Nuclear receptor corepressor N-CoR: role in transcriptional repression2004Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The human body consists of a multitude of cells of varying appearance and function. With a few exceptions they are genetically identical, and the key to their divergence lies in their different specific patterns of gene expression. Gene expression may be regulated at the level of transcription, in two opposing directions; either activation or repression. Gene transcription is controlled by transcription factors, which bind to regulatory DNA sequences, and direct gene expression in concert with auxiliary proteins. Among these the nuclear receptor corepressor N-CoR holds a central position. It serves as a docking unit between many different DNA-bound transcription factors, such as nuclear receptors, and large complexes of repressor proteins. Many repressor complexes of distinct compositions have been shown to contain N-CoR.

    N-CoR plays a vital part in normal fetal development, and its involvement has been implicated in several pathological conditions. It has been shown to interact with unliganded nuclear receptors via CoRNR-box motifs in the C-terminal half of the protein. We have identified an NR-box motif, typical of coactivators, in the N-terminal part of N-CoR, which we have shown to be capable of interacting with the nuclear receptors RARα and TRß both in vitro and in vivo. A mutated NR-box motif did not interact accordingly. We discovered that the NR-box motif found in N-CoR displayed a ligand-dependent interaction with TRß in GST pulldown experiments, and that the immediate NR-box environment in N-CoR resembles NRbox environments in the coactivator CBP. We investigated a possible role for theN-CoR NRbox motif in regulation of the TSHß gene from a negative TR response element found in its promoter. In transient transfectiqns of GH3 cells, we found that both TRß3 and N-CoR are necessary for ligand-induced repression from this response element to occur. Mutating the NR-box abolished the repressive potential of N-CoR. The results were corroborated by results from transient transfections of HEK293/T cells, where siRNA-targeted degradation of endogenous N-CoR mRNA annihilated the ligand-induced repression, and where wild type mouse N-CoR but not mutated N-CoR restored the repression. In vitro binding assays also showed that TR, bound to its negative response element in the TSHß gene promoter, displayed an obligate ligand dependence in its interaction with N-CoR.

    In several different leukemias N-CoR holds a key role. Abberant transcription factors bind stronger toN-CoR than their normal counterparts, leading to constitutive repression of key genes in hematopoiesis. Retinoid signaling, mediated by RARs plays a central part in differentiation of myeloid cells. We therefore investigated the extent of N-CoR expression in the myeloid cell line THP-1 during differentiation. Analyses both at mRNA- and at protein level showed that N-CoR expression was down-regulated as the myeloid cells differentiated. Exploring the effects of this on genes controlled by retinoic acid, we found in transient transfections of THP-1 cells that N-CoR modulated the expression level both at basal and at ligand-activated level. Several reports by others have also emphasized the importance of relative levels of different coregulatory proteins for determining the amplitude of the transcriptional response.

    N-CoR binds both to transcription factors and to repressor complexes, but so far no report has been published regarding its possible DNA-binding capacity, anticipated by analysis of its amino acid sequence. Employing the selected and amplified binding sites (SAAB) assay we showed that N-CoR bound to DNA. Sequence determination resulted in the identification of a DNA sequence, ATNNTNCTC, which binds specifically toN-CoR. This finding adds another variable in the spectrum of N-CoR interactions.

  • 229.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-β gene2005In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 97, no 4, p. 322-327Article in journal (Refereed)
    Abstract [en]

    Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ). Transient transfection experiments now suggest that TRβ and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-β (TSHβ) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRβ and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRβ cooperate in the regulation of the TSHβ gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.

  • 230.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)2004In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 91, no 4-5, p. 191-196Article in journal (Refereed)
    Abstract [en]

    Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor β and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif.

    This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoRLDNLL) fused to GST interacted with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ) in vitro. Similarly, [35S-methionine]N-CoRLDNLL interacted with a RARα fusion protein. N-CoRLDNLL also bound to RARα in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARα in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARα and TRβ.

  • 231.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    N-CoR interacts directly with the DNA sequence ATNNTNCTCManuscript (preprint) (Other academic)
    Abstract [en]

    N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of thyroid and retinoid hormone receptors) are large proteins which are constituent parts of numerous multicomponent repressor complexes. They function by recruiting repressors to the sites of DNA-bound transcription factors, thereby modifying chromatin condensation to restrict access for general transcription factors to a regulated gene. Both N-CoR and SMRT contain two highly conserved SANT (SWI3, ADA2, N-CoR, TFIIIB) domains which, by homology, have been suggested to confer DNA-binding properties. To date however, there have been no data published that support a DNA-binding function for N-CoR (or SMRT). To investigate if N-CoR is capable of binding to DNA, we used stably transfected S2 cells expressing FLAGtagged N-CoR We then allowed N-CoR to interact with double stranded degenerated oligonucleotides. Our results showed that oligonucleotides were retained by N-CoR, and that these contained the consensus sequence ATNNTNCTC.

  • 232.
    Loinder, Kristina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    The nuclear receptor corepressor (N-CoR) modulates basal and activated transcription of genes controlled by retinoic acid2003In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 84, no 1, p. 15-21Article in journal (Refereed)
    Abstract [en]

    Variation in cell morphology and function is caused by differentiation. In myeloid differentiation, retinoid signaling, acting through heterodimers consisting of retinoic acid receptor and retinoid X receptor (RAR/RXR) plays a crucial part. The RAR/RXR heterodimers bind to naturally occurring response elements in the promoter regions of target genes, deciding whether the gene is to be transcribed or not. In the absence of the RAR-specific ligand all trans retinoic acid, RAR/RXR heterodimers are associated with the nuclear receptor corepressor N-CoR or the related SMRT.

    Here we show, using Western, far-Western and Northern blot techniques, that when the human monocytic cell line THP-1 is allowed to differentiate into macrophage-like cells the expression of N-CoR is down-regulated both at the protein and at the mRNA level. To investigate how this affects the transcriptional activity of retinoic acid response element (RARE)-controlled genes, we performed transient transfection experiments in THP-1 and CV-1 cells. The results indicate that N-CoR functions not merely as a repressor of basal transcription, but rather as a modulator of both basal and ligand-activated transcription of genes controlled by RAR/RXR heterodimers in a dose-dependent manner.

  • 233.
    Lundin, Anna-Carin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Eriksson, Birgitta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Bergman-Jungeström, Malin
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Wingren, Sten
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Association of breast cancer progression with a vitamin D receptor gene polymorphism1999In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 59, no 10, p. 2332-2334Article in journal (Refereed)
    Abstract [en]

    The vitamin D3 receptor gene (VDR) contains a TaqI RFLP that is associated with increased VDR mRNA stability, increased serum levels of 1α,25-dihydroxyvitamin D3 (1,25-D3), and decreased risk for prostate cancer. Determination of the TaqI genotype, in a group of young women with breast cancer (n = 111; age, <37 years) and a control population (n = 130), revealed no overall association to risk for breast cancer. However, patients without TaqI site (TT genotype) showed a significantly increased risk for lymph node metastasis (relative risk, 1.8, 95% confidence interval, 1.3- 2.6). Furthermore, a tendency toward an increased survival was found among estrogen receptor-positive, tamoxifen-treated patients who were homozygous for the TaqI site (P = 0.075). We conclude that polymorphism in the VDR gene may influence tumor progression and tamoxifen treatment response in early- onset breast carcinomas.

  • 234.
    Lundmark, Katarzyna
    et al.
    Division of Pathology, Karolinska University Hospital, Huddinge, Sweden.
    Westermark, Gunilla
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Olsén, Arne
    Department of Clinical Immunology, Göteborg University, Göteborg, Sweden.
    Westermark, Per
    Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Protein fibrils in nature can enhance amyloid protein A amyloidosis in mice: Cross-seeding as a disease mechanism2005In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 102, no 17, p. 6098-6102Article in journal (Refereed)
    Abstract [en]

    Secondary, or amyloid protein A (AA), amyloidosis is a complication of chronic inflammatory diseases, both infectious and noninfectious. AA constitutes the insoluble fibrils, which are deposited in different organs, and is a major N-terminal part of the acute phase protein serum AA. It is not known why only some patients with chronic inflammation develop AA amyloidosis. Nucleation is a widely accepted mechanism in amyloidogenesis. Preformed amyloid-like fibrils act as nuclei in amyloid fibril formation in vitro, and AA amyloid fibrils and synthetic amyloid-like fibrils also may serve as seed for fibril formation in vivo. In addition to amyloid fibrils, there is a variety of similar nonmammalian protein fibrils with β-pleated structure in nature. We studied three such naturally occurring protein fibrils: silk from Bombyx mori, Sup35 from Saccharomyces cerevisiae, and curli from Escherichia coli. Our results show that these protein fibrils exert amyloid-accelerating properties in the murine experimental AA amyloidosis, suggesting that such environment factors may be important risk factors in amyloidogenesis.

  • 235.
    Lundmark, Katarzyna
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla T.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lindquist, Susan
    Howard Hughes Medical Institute, University of Chicago, Il, USA.
    Westermark, Per
    Department of Genetics and Pathology, Uppsala University, Sweden.
    Naturally occurring fibrillar proteins can induce AA amyloidosis by a prion-like mechanismManuscript (preprint) (Other academic)
    Abstract [en]

    Experimental AA amyloidosis, where the acute phase protein serum AA (SAA) forms amyloid fibrils, can be induced in mice provoked with inflannnatmy challenge. The time for development of amyloid is dramatically shortened when the animals concomitantly receive extract of a tissue from another mouse with amyloid 1-3. The active elusive principle has been named Amyloid Enhancing Factor (AEF) and experimental secondary amyloidosis was supposed to be a nucleation dependent process. The nature of the nucleus, however, was unknown for a long time. Our studies with synthetic amyloid-like fibrils made frmn short amyloidogenic pep tides instead of AEF 4, 5, indicate that the amyloid fibrils theruselves may act as nuclei for fibril formation (Fig. 1a). Here we present the enhanced development of AA -amyloidosis by naturally occurring amyloid-like protein fibrils.

  • 236.
    Lutz, Barbara
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Structural and functional regeneration of muscle-related axons after transection and repair of the rat sciatic nerve using nonvascularized autologous fascia as a barrier between tibial and peroneal nerve fascicles2004In: Journal of reconstructive microsurgery, ISSN 0743-684X, E-ISSN 1098-8947, Vol. 20, no 8, p. 637-644Article in journal (Refereed)
    Abstract [en]

    Aberrant reinnervation of target organs caused by misdirected axonal growth at the repair site is a major reason for the poor functional outcome usually seen after peripheral nerve transection and repair. This study investigates whether the criss-crossing of regenerating rat sciatic nerve axons between tibial and peroneal nerve fascicles can be reduced by using non-vascularized autologous fascia as a barrier.

    The left sciatic nerve was transected and repaired at midthigh as follows: epineurialy sutures (Group A); fascicular repair of tibial and peroneal nerve fascicles (Group B); fascicular repair of tibial and peroneal nerve fascicles separating the two fascicles by non-vascularized autologous fascia (Group C). In the control Group D, only the left tibial fascicle was transected and repaired. Five months postoperatively, the outcome of regeneration was evaluated by histology, by retrograde tracing, and by assessment of the contraction force of the gastrocnemius and tibial anterior muscles. The tracing experiments showed that muscle reinnervation was less abnormal in Group C than in Groups A and B. However, muscle contraction force was not better in Group C than in Groups A and B. With respect to the peroneal nerve innervated muscle, the contraction force in Group C was significantly lower than in Group B. The histologic picture indicated that this inferior result in Group C was due to nerve compression caused by fibrotic scar tissue at the site of the fascia graft.

    Results of this study show that a non-vascularized autologous fascial graft used as a barrier between two sutured nerve fascicles in adjacency reduces criss-crossing of regenerating axons between the fascicles, but causes significant nerve compression.

  • 237.
    Löfgren, Ragnhild
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Phagocytic-receptor signaling in human neutrophils1999Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The neutrophil granulocyte is one of the most mobile cells in the body and through a number of functions represents the first line of defense against invading pathogens. Adhesion and chemotactic receptors on the cell surface work together through modulation of the cytoskeleton, thereby enabling cell movement. Engulfment is facilitated by opsonization of the microbes with C3b or C3bi complement fragments or with immunoglobulins, proteins that respectively bind to complement receptors (CR) and Fey receptors (FcyR) on the neutrophil. The aim of the present study was to investigate the signal transduction properties of CR and FcyR on human neutrophils. In particular, the investigations focused on second messengers of importance for regulating actin polymerization and NADPH-oxidase activity. This was approached by selective activation of each receptor on non-adherent human neutrophils using specific antibodies that were either cross-linked or prefixed on bacterial particles.

    Both stimulation with f.MetLeuPhe and engagement of CR3 caused an increase in filamentous actin (F-actin) and activation of phosphatidylinositol 3-klnase with the subsequent formation of phosphatidylinositol trisphosphate (PIP,). We found that the F-actin response mediated by fMetLenPhe and CR3 were different. The F-actin response induced by fMetLeuPhe declined rapidly whereas the response induced by engagement of CR3 was more sustained. This is hypothesized to be due to the inability of CR3 to induce a cAMP signal, since direct addition of cAMP and 1-isobutyl-methylxantine (IBMX, a phosphodiesterase inhibitor) to electropermeabilized neutrophils caused a prompt reversal of the CR3-induced F-actin elevation.

    Engagement of CR3 and CR1 by antibody cross-linking induced a Ca2+ signal and phospholipase D (PLD) activation. Furthermore, the PLD response was potentiated by PMA pretreatment and was dependent on the form of ligand presentation. Both FcyR (FcyRITA and FcyRIIIB) and CR3 induced activation of NADPH-oxidase, a response that was dependent on intracellular Ca2+, tyrosine kinase activation and PLD activity. However, only FcyRllA induced a strong phosphorylation of p72''\ indicating that CR3 and FcyRliA might use different pathways leading to NADPH-oxidase activation.

  • 238.
    Löfgren, Ragnhild
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Signaling capacity of ß2-integrins in relations to neutrophil motility1996Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    The neutrophil granulocyte is one of the most mobile cell types in the human body and represents the first line of defense against invading pathogens. Adhesion and chemotactic receptors on the cell surface work together through modulations of the cytoskeleton and thereby cause the cell to move. Cell movement is dependent on actin reorganization but our understanding of the molecular mechanisms behind this is limited. The aim of the present thesis was to investigate the signaling capacity of the ß2-integrins and to try to define a signaling event responsible for the regulation of actin assembly in human neutrophils.

    Stimulation with the chemotactic peptide fMetLeuPhe causes activation of a pertussis toxin-sensitive GTP-binding protein (G-protein) that in turn triggers a cascade of events in the human neutrophil. fMetLeuPhe stimulates an increase in filamentous actin (F-actin) and activation of the phosphatidylinositol 3-kinase (PI3-kinase) and the subsequent formation of phosphatidylinositol trisphosphate (PIP3). A role for PIP3 in promoting actin assembly has been suggested since the time course for PIP3 formation directly correlates with actin polymerization. The present study shows that engagement of ß2-integrins by antibody cross-linking induced a calcium signal and actin polymerization, but these responses, however, were not sensitive for pertussis toxin. The F-actin response induced by fMetLeuPhe was rapidly declining whereas the response induced by engagement of ß2-integrins was more sustained. This can be due to the inability of ß2-integrins to induce a cAMP signal since direct addition of cAMP and 1-isobutyl-methylxantine (IBMX, a phophodiesterase inhibitor) to electropermeabilized neutrophils caused a prompt reversal of the ß2-integrininduced F-actin elevation. The signaling capacity of the ß2-integrins was positively modulated by pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA, a protein kinase C activating agent). Priming neutrophils with lnM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the ß2-integrin-induced F-actin response. Interestingly, engagement of ß2-integrins was shown to induce formation of PIP3 a fmding that further supports the suggested role for PIP3 in promoting actin polymerization.

  • 239.
    Löfgren, Ragnhild
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Serrander, Lena
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Forsberg, Maria
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Wilsson, Åsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Wasteson, Åke
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    CR3, FcγRIIA and FcγRIIIB induce activation of the respiratory burst in human neutrophils: the role of intracellular Ca2+, phospholipase D and tyrosine phosphorylation1999In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1452, no 1, p. 46-59Article in journal (Refereed)
    Abstract [en]

    Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcγRIIA and FcγRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcγRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcγRIIA Pansorbins induced an even weaker signal. However, FcγRIIA induced strong phosphorylation of p72syk whereas FcγRIIIB induced only a very weak p72syk phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72syk was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72syk that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72syk phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcγRIIA and FcγRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72syk is an early signal in the cascade induced by FcγRIIA but not by CR3.

  • 240.
    Ma, Zhi
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sakagashira, S.
    First Department of Medicine, Wakayama University of Medical Science, Kagawa Medical University, Japan.
    Sanke, T.
    First Department of Medicine, Wakayama University of Medical Science, Kagawa Medical University, Japan.
    Gustavsson, Å
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Sakamoto, H.
    Department of Pathology, Kagawa Medical University, Japan.
    Engstrom, U.
    Ludwig Institute of Cancer Research, Uppsala Branch, Uppsala University, Uppsala, Sweden.
    Nanjo, K.
    First Department of Medicine, Wakayama University of Medical Science, Kagawa Medical University, Japan.
    Westermark, P.
    Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
    Enhanced in vitro production of amyloid-like fibrils from mutant (S20G) islet amyloid polypeptide2001In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 8, no 4, p. 242-249Article in journal (Refereed)
    Abstract [en]

    Islet amyloid polypeptide (IAPP, “amylin”) is the amyloid-fibril-forming polypeptide in the islets of Langerhans associated with type 2 diabetes mellitus. A missense mutation in the IAPP gene associated with early-onset type 2 diabetes has been identified in the Japanese population. This mutation results in a glycine for serine substitution at position 20 of the mature IAPP molecule. Whether or not formation of islet amyloid with resulting destruction of islet tissue is the cause of this diabetes is yet not known. The present in vitro study was performed in order to investigate any influence of the amino acid substitution on the fibril formation capacity. Synthetic full-length wild type (lAPPwt) and mutant (IAPPS20G) as well as corresponding truncated peptides (position 18-29) were dissolved in dimethylsulfoxide (DMSO) or in 10% acetic acid at a concentration of 10 mg/mL and their fibril forming capacity was checked by Congo red staining, electron microscopy, a Congo red affinity assay and Thioflavine T fluorometric assay. It was found that full-length and truncated IAPPS20G both formed more amyloid-like fibrils and did this faster compared to IAPPwt. The fibril morphology differed slightly between the preparations. Conclusion: The amino acid substitution (S20G) is situated close to the region of the IAPP molecule implicated in the IAPP fibrillogenesis. The significantly increased formation of amyloid-like fibrils by IAPPS20G is highly interesting and may be associated with an increased islet amyloid formation in vivo and of fundamental importance in the pathogenesis of this specific form of diabetes.

  • 241.
    Ma, Zhi
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla T.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Effects of free fatty acid on polymerization of islet amyloid polypeptide (IAPP) in vitro and on amyloid fibril formation in cultivated isolated islets of transgenic mice overexpressing human IAPP2002In: Molecular medicine (Cambridge, Mass. Print), ISSN 1076-1551, E-ISSN 1528-3658, Vol. 8, no 12, p. 863-868Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Islet amyloid polypeptide (IAPP) is deposited as amyloid in the islets of Langerhans in type 2 diabetes. The mechanism behind the formation of the cytotoxic fibrils is unknown. Islet amyloid develops in a mouse IAPP null mouse strain that expresses human IAPP (+hIAPP/-mIAPP) after 9 months on a high-fat diet. Herein we investigate the effect that individual free fatty acids (FFAs) exert on formation of amyloid-like fibrils from synthetic IAPP and the effects of FFAs on IAPP polymerization in +hIAPP/-mIAPP islets cultivated in vitro.

    MATERIALS AND METHODS: In the study myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid were used together with albumin. Thioflavin T (Th T) assay was used for quantification of amyloid-like fibrils. Islets were isolated from the +hIAPP/-mIAPP transgenic strain and cultured in the presence of the FFAs for 2 days. Immuno-electron microscopy was used for evaluation.

    RESULTS: The Th T assay showed that all studied FFAs potentiated fibril formation but that myristic acid revealed the highest capacity. In some cells from cultured islets, intragranular aggregates were present. These aggregates had a filamentous appearance and labeled with antibodies against IAPP. In some cells cultured in the presence of linoleic acid, large amounts of intracellular amyloid were present. Earlier, this has not been observed after such a short incubation period.

    CONCLUSIONS: Our studies suggest that FFAs can potentiate amyloid formation in vitro, probably without being integrated in the fibril. Cultivation of +hIAPP/-mIAPP transgenic mouse islets with FFAs results in altered morphology of the secretory granules with appearance of IAPP- immunoreactive fibrillar material. We suggest that such fibrillar material may seed extracellular amyloid formation after exocytosis.

  • 242.
    Ma, Zhi
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Westermark, P.
    Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Linköping University, Faculty of Health Sciences.
    Westermark, Gunilla
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Amyloid in Human Islets of Langerhans: Immunologic Evidence That Islet Amyloid Polypeptide Is Modified in Amyloidogenesis2000In: Pancreas, ISSN 0885-3177, E-ISSN 1536-4828, Vol. 21, no 2, p. 212-218Article in journal (Refereed)
    Abstract [en]

    Amyloid derived from the beta-cell product islet amyloid polypeptide (IAPP) has been implicated for a beta-cell lesion in Type II diabetes mellitus. The pathogenesis of islet amyloid is poorly understood, and in addition to an amyloidogenic IAPP molecule and possibly increased concentration of IAPP, other unknown factors seem to be included. It was shown previously that polyclonal rabbit IAPP antisera label beta cells close to amyloid only weakly. Whether this lack of immunoreactivity depends on lack of IAPP or on hidden epitopes is in question. In the present study, we show that the IAPP immunoreactivity of these beta cells is possible to retrieve. On the other hand, the monoclonal IAPP antibody 4A5, which labels IAPP in beta cells, does not label IAPP in its native amyloid form. We show evidence that this lack of immunoreactivity is not dependent on conformational change of the IAPP molecules in the amyloidogenesis but is likely owing to glycation of IAPP in human islet amyloid deposits.

  • 243.
    Magnusson, Anna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Central vestibular compensation: the role of the GABAB receptor2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The remarkable capacity for adaptive plastic changes in response to changed internal or external conditions is a distinctive feature of the vestibular system. Even in adults the system can be modified throughout life due to altered conditions caused by disease. trauma, medical treatment or normal ageing. Central nervous plastic changes following a unilateral peripheral vestibular loss are summarised by the term 'vestibular compensation'. This concept has become the most extensively investigated experimental model in studies of vestibular plasticity. The vestibular system governs a number of reflexes of which one is maintaining a stable gaze when the head moves - the vestibuloocular reflex. Since this reflex is relatively easy to quantify with non-invasive methods it constitutes an excellent tool for studying vestibular function in health and disease. Furthermore, the underlying neuronal circuitry of the reflex is phylogenetically ancient.

    γ-Aminobutyric acid (GABA) is the most widely distributed inhibitory neurotransmitter in the vertebrate central nervous system. It acts via the classical GABAA and the more recently discovered GABAB receptors, the physiological functions of which are just beginning to emerge. The studies that provide the basis for this thesis systematically investigate the functional significance of the GABAB receptors for vestibular compensation during several stages after unilateral vestibular loss in rats. Firstly, the long-term maintenance of the partially normalised vestibular function weeks- months after the sensory loss was investigated (I and 11). Subsequently, the compensation that normalises the function of the vestibular system within a few days after the loss was investigated (III). Finally, in order to be able to investigate the acute stage, minutes - hours after unilateral vestibular loss, a method for reversible inactivation of the vestibular sensory input was developed (IV). In addition to information about the role of GABAB receptor function during this stage. the method also revealed the immediate behavioural consequences following a sudden transient vestibular loss as well as compensatory modulations that outlasted the inactivation of the sensory input (IV).

    In summary, this thesis demonstrates a concrete physiological role of the GABAB receptors in a well-characterised neural system related to a specific behaviour. A direct causal relationship between the GABAB receptors and the physiological changes underlying compensation from a unilateral peripheral vestibular loss is established for all stages of the compensatory process. The physiological effect is partly mediated through an endogenous tonic control of the receptor. Furthermore, this thesis elucidates the immediate behavioural consequences of an acute transient loss of sensory vestibular input.

  • 244.
    Magnusson, Anna K.
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Eriksson, Birgitta
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Tham, Richard
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Effects of the GABA agonists baclofen and THIP on long-term compensation in hemilabyrinthectomised rats1998In: Brain Research, ISSN 0006-8993, E-ISSN 1872-6240, Vol. 795, no 1-2, p. 307-311Article in journal (Refereed)
    Abstract [en]

    Horizontal eye movements, elicited by sinusoidal rotation in darkness, were recorded with a magnetic search coil technique in pigmented rats, hemilabyrinthectomised 8–12 weeks before the investigation. Separate gains during rotation towards the lesioned side (LS) and the intact side (IS) were calculated by a computer program, demonstrating an asymmetry. Systemic single administration of the GABAB agonist baclofen caused a dose-related temporary rebalancing of the compensatory eye movements to the LS and the IS. At an optimal dose of 14 μmol/kg b.wt symmetry was achieved by excitation of eye movements during rotation to the LS and depression during rotation to the IS. Administration of the GABAA agonist THIP did not obviously reduce the asymmetry. It is suggested that stimulation of GABAB receptors modifies the tonic imbalance between the bilateral vestibular nuclei and/or the central processing of the input from the peripheral sensory organs.

  • 245.
    Magnusson, Anna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lindström, Sivert
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Tham, Richard
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    GABAB receptors contribute to vestibular compensation after unilateral labyrinthectomy in pigmented rats2000In: Experimental Brain Research, ISSN 0014-4819, E-ISSN 1432-1106, Vol. 134, no 1, p. 32-41Article in journal (Refereed)
    Abstract [en]

    The horizontal vestibulo-ocular reflex was studied in pigmented rats, which had been unilaterally, chemically labyrinthectomised 6–144 days previously. During this partially compensated stage after unilateral labyrinthectomy (UL), both static and dynamic deficits remain. The former was evaluated by recording of spontaneous eye movements in darkness, and the latter by estimating the slow-phase velocity (SPV) gain of compensatory eye movements during horizontal vestibular stimulation. The GABAB agonist baclofen caused a reversal of the remaining ipsilesional drift of the eyes in darkness into a nystagmus with a contralesional slow phase. The GABAB antagonist CGP 36742 caused a decompensation by exaggerating the remaining ipsilesional eye drift. Further, baclofen equilibrated or reversed the asymmetry between ipsi- and contralesional SPV gains during horizontal sinusoidal rotations at 0.2 Hz and 0.8 Hz. This was achieved by an increase in the ipsilesional gain and a decrease in the contralesional gain. The phase lead during sinusoidal rotation (0.2 Hz) was larger following rotation to the lesioned side than to the intact side in UL rats. This asymmetry was reversed by baclofen. CGP 36742 inhibited the effects of baclofen, while the antagonist per se aggravated SPV gain and phase lead asymmetries in UL rats during vestibular stimulation. Per- and post-rotatory nystagmus induced by velocity step stimulation revealed an imperfect velocity-storage function in UL animals, which was modulated by baclofen. An investigation of the baclofen effect on SPV gain asymmetry during different time intervals after chemical UL showed a completely developed effect on the 6th day. Bilateral flocculectomy did not alter the effects of baclofen on UL animals. It is concluded that physiological stimulation of GABAB receptors contributes to minimise the vestibulo-oculomotor asymmetry during the partially compensated period after UL. Administration of an agonist or an antagonist changes the asymmetry towards the ipsi- or contralesional side, possibly by altering the spontaneous neuronal activity in the bilateral medial vestibular nuclei. The results are compatible with a hypothesis, supported by in vitro slice experiments, that the efficacy of GABAB receptors is up-regulated on the ipsilesional side and down-regulated on the contralesional side.

  • 246.
    Magnusson, Anna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Tham, Richard
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Vestibulo-oculomotor behaviour in rats following a transient unilateral vestibular loss induced by lidocaine2003In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 120, no 4, p. 1105-1114Article in journal (Refereed)
    Abstract [en]

    The effects of a transient vestibular nerve blockade, achieved by intra-tympanic instillation of lidocaine, were studied in rats by recording horizontal eye movements in darkness. Evaluation of the dose-response relationship showed that a maximal effect was attained with a concentration of 4% lidocaine. Within 15 min of lidocaine instillation, a vigorous spontaneous nystagmus was observed which reached maximal frequency and velocity of the slow phase after about 20 min. Subsequently, the nystagmus failed for approximately half an hour before it reappeared. This could be avoided by providing visual feedback in between the recordings in darkness or by a contralateral instillation of 2.5% lidocaine. It is suggested that the failure reflects an overload of the vestibulo-oculomotor circuits.

    After recovery from the nerve blockade, when the gaze was stable, dynamic vestibular tests were performed. They revealed that a decrease of the slow phase velocity gain and the dominant time constant during, respectively, sinusoidal- and step stimulation toward the unanaesthetised side, had developed with the nerve blockade. These modulations were impaired by a nodulo-uvulectomy but not by bilateral flocculectomy, which is consistent with the concept of vestibular habituation.

    A GABAB receptor antagonist, CGP 56433A, given systemically during the nerve blockade, aggravated the vestibular asymmetry. The same effect has previously been demonstrated in both short- (days) and long-term (months) compensated rats, by antagonising the GABAB receptor.

    In summary, this study provides the first observations of vestibulo-oculomotor disturbances during the first hour after a rapid and transient unilateral vestibular loss in the rat. By using this method, it is possible to study immediate behavioural consequences and possible neural changes that might outlast the nerve blockade.

  • 247.
    Magnusson, Anna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ulfendahl, M.
    Institute for Hearing and Communication Research and Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Tham, Richard
    Linköping University, Department of Neuroscience and Locomotion, Oto-Rhiono-Laryngology and Head & Neck Surgery. Linköping University, Faculty of Health Sciences.
    Early compensation of vestibulo-oculomotor symptoms after unilateral vestibular loss in rats is related to GABAB receptor function2002In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 111, no 3, p. 625-634Article in journal (Refereed)
    Abstract [en]

    The horizontal vestibulo-oculomotor reflex was studied in pigmented rats during the first 5 days after a unilateral chemical or surgical vestibular deafferentation. Spontaneous eye movements in darkness and slow phase velocity gain of compensatory eye movements during horizontal sinusoidal rotation were evaluated. The most evident vestibulo-oculomotor symptom immediately after a unilateral vestibular loss was a spontaneous nystagmus, which gradually abated during the following days. Further, an asymmetry between ipsi- and contra-lesional gains was evident during sinusoidal vestibular stimulation. Single systemic doses of the GABAB receptor antagonist [3-[1-(S)-[[3-(cyclohexylmethyl)-hydroxyphosphinoyl]-2-(S)-hydroxypropyl]amino]ethyl]-benzoic acid (CGP 56433A), the agonist baclofen, or the GABAA receptor agonist (4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridin-3-ol (THIP) were given at different intervals after unilateral vestibular deafferentation. CGP 56433A highly aggravated the vestibulo-oculomotor symptoms, observed as an increase in spontaneous nystagmus and slow phase velocity gain asymmetry. This effect was most pronounced during the first 2 days after unilateral vestibular loss, when CGP 56433A even decompensated the vestibular system to the extent that all vestibular responses were abolished. Baclofen caused no effect during the first days after unilateral vestibular loss, but in parallel with the abatement of spontaneous nystagmus, the drug equilibrated or even reversed the remaining spontaneous nystagmus with corresponding effects on the slow-phase velocity gain asymmetry. The effects of baclofen were very similar after both chemical and surgical deafferentation. THIP caused a slight depression of all vestibular responses. All single dose effects of the drugs were transient.

    Altogether these results reveal that endogenous stimulation of GABAB receptors in GABA-ergic vestibulo-oculomotor circuits are important for reducing the vestibular asymmetry during the early period after unilateral vestibular deafferentation. A possible role for GABAB receptors in the reciprocal inhibitory commissural pathways in the vestibular nuclei is suggested.

  • 248. Mamedov, Fikret
    et al.
    Rintamäki, Eevi
    Aro, Eva-Mari
    Andersson, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Styring, Stenbjörn
    Influence of protein phosphorylation on the electron-transport properties of Photosystem II2002In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 74, no 1, p. 61-72Article in journal (Refereed)
    Abstract [en]

    Many of the core proteins in Photosystem II (PS II) undergo reversible phosphorylation. It is known that protein phosphorylation controls the repair cycle of Photosystem II. However, it is not known how protein phosphorylation affects the partial electron transport reactions in PS II. Here we have applied variable fluorescence measurements and EPR spectroscopy to probe the status of the quinone acceptors, the Mn cluster and other electron transfer components in PS II with controlled levels of protein phosphorylation. Protein phosphorylation was induced in vivo by varying illumination regimes. The phosphorylation level of the D1 protein varied from 10 to 58% in PS II membranes isolated from pre-illuminated spinach leaves. The oxygen evolution and QA- to QB(QB-) electron transfer measured by flash-induced fluorescence decay remained similar in all samples studied. Similar measurements in the presence of DCMU, which reports on the status of the donor side in PS II, also indicated that the integrity of the oxygen-evolving complex was preserved in PS II with different levels of D1 protein phosphorylation. With EPR spectroscopy we examined individual redox cofactors in PS II. Both the maximal amplitude of the charge separation reaction (measured as photo-accumulated pheophytin-) and the EPR signal from the QA- Fe2+ complex were unaffected by the phosphorylation of the D1 protein, indicating that the acceptor side of PS II was not modified. Also the shape of the S2 state multiline signal was similar, suggesting that the structure of the Mn-cluster in Photosystem II did not change. However, the amplitude of the S2 multiline signal was reduced by 35% in PS II, where 58% of the D1 protein was phosphorylated, as compared to the S2 multiline in PS II, where only 10% of the D1 protein was phosphorylated. In addition, the fraction of low potential Cyt b559 was twice as high in phosphorylated PS II. Implications from these findings, were precise quantification of D1 protein phosphorylation is, for the first time, combined with high-resolution biophysical measurements, are discussed.

  • 249. McAdam, KPWJ
    et al.
    Raynes, JG
    Alpers, MP
    Westermark, Gunilla
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Westermark, P
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Molecular and Immunological Pathology. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pathology and Clinical Genetics.
    Amyloidosis: a global problem common in Papua New Guinea. 1999In: Papau New Guinea Medical Journal, ISSN 0031-1480, Vol. 39, p. 284-296Article in journal (Refereed)
  • 250.
    Metcalf, Kerstin
    et al.
    Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
    Berg, Anna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ericson, Ann-Charlott
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lisander, Björn
    Linköping University, Department of Medicine and Care, Anaesthesiology. Linköping University, Faculty of Health Sciences.
    Nitric oxide does not cause extravasation in endotoxemic rats2005In: Journal of Trauma, ISSN 0022-5282, E-ISSN 1529-8809, Vol. 58, no 5, p. 1047-1054Article in journal (Refereed)
    Abstract [en]

    Background: Nitric oxide (NO) formed from inducible NO synthase (iNOS) is assumed to promote vascular permeability in sepsis and endotoxemia.

    Methods: Thirty-seven anesthetized rats were examined for the effects of endotoxin. After randomization, 17 animals had lipopolysaccharide (LPS) administered and 20 rats served as controls and were given the corresponding volume of saline. The observation period was 5 hours after administration of endotoxin. Mean arterial blood pressure, heart rate, and hematocrit were recorded in all animals, and transcapillary exchange of albumin, tissue water content, immunohistochemistry for nitric oxide synthase, and blood gases were investigated in subsets of animals.

    Results: When anesthetized rats were studied for 5 hours after endotoxin (LPS), the sequestration of albumin decreased in the intestine (double-isotope method) and there was no increased water content (freeze-drying technique) when the elevated tissue plasma volume of the LPS-treated rats was corrected for. Immunohistochemical methods showed a similar distribution and intensity of staining for endothelial NOS and neuronal NOS in LPS and control groups. In the lung of the LPS-treated rats, there was a significantly larger number of infiltrating, inflammatory cells staining for iNOS. There was no iNOS demonstrated in vascular structures or heart.

    Conclusion: At 5 hours after LPS, there was no increased loss of water or albumin from the circulation. This challenges the notion that NO causes vascular damage in endotoxemia and extravasation as an obligatory sequela to endotoxemia.

2345678 201 - 250 of 355
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