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  • 101.
    Esbjörner, Elisabeth
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Studies on albumin binding properties in pregnancy and early infancy: with special reference to maternal sulphasalazine treatment1990Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Sulphasalazine (SASP) has been avoided during the later part of pregnancy and during breast-feeding as some sulphonamides possess the ability to displace bilirubin from albumin and thus increase the risk of bilirubin-induced brain damage in jaundiced neonates. However, withdrawal of SASP in a woman with ulcerative colitis would mean a cqnsiderable risk of relapse of her disease.

    In this study, SASP and its metabolite sulphapyridine (SP) was shown to pass the placenta. Sulphapyridine but not SASP appeared in breast-milk, although concentrations in breast-fed infants were low. The substances were eliminated slower in newborns than in adults.

    The possible bilirubin-displacing effect of SASP and SP was evaluated by using the MADDS (monoacetyldiaminodiphenyl sulphone) method to determine the binding 'properties of serum albumin.

    MADDS is used as a deputy ligand for bilirubin. In vivo and in vitro studies, using the MADDS and the peroxidase methods, showed that SASP and SP in pharmacological concentrations did not displace bilirubin from albumin.

    During that study it was noted that the reserve albumin concentration for MADDS was far lower in women at delivery than in non-pregnant women. In a longitudinally followed group of pregnant women, the reserve albumin concentration was gradually lowered during pregnancy, reaching 530/o of the concentration in non-pregnant women at term. This can have pharmacokinetic effects on those drugs that share the binding function on albumin with MADDS and bilirubin. The reduction of the reserve albumin concentration was due to a reduced albumin concentration during pregnancy but also to a reduced binding ability of the albumin molecule.

  • 102.
    Evertsson, Sofia
    et al.
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Bartik, Zsuzsa
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Zhang, Hong
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Jansson, Agneta
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Sun, Xiao-Feng
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Apoptosis in relation to proliferating cell nuclear antigen and Dukes' stage in colorectal adenocarcinoma1999In: International Journal of Oncology, ISSN 1019-6439, Vol. 15, no 1, p. 53-58Article in journal (Refereed)
    Abstract [en]

    Colorectal cancer is a disease that is associated with default in the balance of apoptotic regulation. In the present study apoptosis was examined in 158 colorectal adenocarcinomas using the terminal deoxynucleotidyl transferase mediated digoxigenin nick end labeling (TUNEL) method. The median apoptotic index (AI) was 0.95% (range 0-6. 68%). Eighty-two tumours exhibited AI 0.95%. We revealed a positive correlation between apoptosis and proliferation determined as the expression of proliferating cell nuclear antigen (PCNA, p=0.002). The frequency of apoptosis increased from Dukes' stage A, B, C to D (p=0.01). No correlations were found between apoptosis and the patients' sex, age, tumour location, growth pattern, differentiation, prognosis, bcl-2, p53 or K-ras. Our findings suggest that we should further investigate the relationship between apoptosis and cellular proliferative activity in colorectal cancer to evaluate whether this might provide additional information in the selection of patients for effective adjuvant therapy.

  • 103. Feng, Wang
    et al.
    Adrian, TE
    Westermark, Gunilla
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Ding, X
    Gasslander, Thomas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Surgery. Östergötlands Läns Landsting, MKC-2, GE: Gastrokir.
    Permert, Johan
    Islet amyloid polypeptide tonally inhiits beta-, alpha-, and delta-cell secretion in isolated rat pancreatic islets.1999In: American journal of physiology, ISSN 0002-9513, Vol. 276, p. 19-24Article in journal (Refereed)
  • 104. Feng, Wang
    et al.
    Westermark, Gunilla
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Gasslander, Thomas
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Surgery. Östergötlands Läns Landsting, MKC-2, GE: Gastrokir.
    Permert, Johan
    Effect of islet amyloid polypeptide on somatostatin inhibition of insulin secretion from isolated rat pancreatic islets.1999In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 72, p. 61-67Article in journal (Refereed)
  • 105.
    Fjellstedt, Erik
    Linköping University, Department of Biomedicine and Surgery, Urology. Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Clinical and genetic studies on patients with cystinuria2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Cystinuria is a genetic disorder with autosomal recessive inheritance. It is caused by a defective proximal tubular reabsorbtion of cystine and the dibasic amino acids. The low urinary solubility of cystine causes a life-long stone disease, contributing to about 1% of all urinary stones in adults. Treatment is based on high fluid intake and the use of sulfhydryl compounds such as tiopronin and D-penicillamine to decrease the urinary concentration of cystine, and alkalinization of the urine to increase the urinary solubility of cystine. Reduction of sodium intake is also a favoured regimen because of eo-transportation of sodium and cystine in the proximal tubules. Advancements in molecular genetics have led to the identification of two genes associated with cystinuria (SLC3A1 and SLC7A9). These genes cannot, however, explain all cases of cystinuria.

    Investigation of SLC3A1 in 53 Swedish patients with cystinuria revealed 12 novel mutations, and the allelic frequency of the most common mutation (M467T) was shown to be 0.5% in a normal population. (Paper I). Studies of SLC7A9 revealed three novel and one previously known mutation. One patient had novel mutations in both alleles, one patient showed a novel mutation in one of the alleles and one patient showed a previously known mutation in SLC7A9 and two in SLC3A1, leaving 14 patients in whom cystinuria was not explained by genetic observations (Paper m. In order to relate these genetic findings to excretion of cystine in the urine, 33 patients treated with sulfhydryl compounds were studied (Paper III). Ten of these patients showed either mutation in one of the SLC3A1 alleles (8) or a complete lack of mutations in both genes (2). These 10 patients showed a significantly higher urinary excretion of total cystine compared to 23 patients in whom both SLC3A1 alleles were mutated (p < 0.01). The same was true for the 8 patients with only one SLC3A1 allele mutated (p < 0.05). These findings support the existence of yet unknown genes involved in the regulation of urinary cystine excretion, the basis of cystine stone formation. Treatment is primarily aimed at the prevention of such stones and should be guided by the urinary cystine concentration, trying to avoid supersaturation. In order to improve patient surveillance in terms of urinary supersaturation with cystine a procedure was introduced comprising one daytime and one night urine sample during the 24-hour period (Paper IV). Twenty-six patients were followed over a 3.5 year period using this strategy. It was found that 47% of cystine supersaturation episodes (> 1200 µmol/L) would have evaded detection by analysis carried out in 24-hour urine collections. Furthermore, a significant decrease in the frequency of renal stone episodes (p < 0.05) and active stone removals (p < 0.01) was found when compared to a previous, equivalent period during which 24-hour urine collections were used. The period guided by divided urine samples was also characterized by a significant decrease in free cystine concentrations (p < 0.01) and a significant increase in urinary volumes (p < 0.05). In the tiopronin-treated patients, there was a significant increase in the tiopronin dose and a subsequent decrease in urinary cystine excretion (p < 0.05). The use of cystine analysis in divided urine samples thus made a higher degree of individual treatment possible. The effects of sodium bicarbonate and potassium citrate were compared in 14 cystinuric patients (Paper V). Potassium citrate has been the favoured agent, as it does not contain sodium, but there have been no reports in which potassium citrate has been compared to sodium bicarbonate in the treatment of patients with cystinuria. Sodium bicarbonate was effective in alkalizing the urine, but caused a significantly increased urinary sodium excretion (p < 0.01). A significant correlation was found between urinary sodium and cystine excretion in tiopronin treated patients (p < 0.001). Potassium citrate was shown to produce a significant increase in urinary pH. Potassium citrate was associated with a significant increase in plasma potassium (p < 0.05), but no case of severe hyperkalemia was found. Potassium citrate could thus be recommended for urinary alkalinization in cystinuric patients without severe renal impairment.

  • 106.
    Forsberg, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Blomgran, Robert
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Lem, Maria
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sebti, Said M.
    Drug Discovery Program, H. Lee Moffitt Cancer Center & Research Institute, Department of Oncology, University of South Florida, Tampa.
    Hamilton, Andrew
    Department of Chemistry, Yale University, New Haven, Connecticut .
    Stendahl, Olle
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Zheng, Limin
    Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages: potential role of Rho–GTPases and Akt2003In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, p. 620-629Article in journal (Refereed)
    Abstract [en]

    In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.

  • 107.
    Forsberg, Maria
    et al.
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Druid, Pia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Zheng, Limin
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Stendahl, Olle
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
    Särndahl, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils2003In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 74, no 4, p. 611-619Article in journal (Refereed)
    Abstract [en]

    Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcγR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcγR and CR3- and FcγR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcγRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.

  • 108.
    Fransén, Karin
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Molecular genetic aspects of colorectal cancer development2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Colorectal cancer (CRC) is one of the most common cancer diseases in the world after lung and female breast cancer and approximately 945 000 new cases are diagnosed every year. CRC is caused by genetic alterations in the DNA, which results in cell cycle acceleration, escape from apoptosis, senescence, angiogenesis, invasion and metastasis. In this thesis, we have investigated molecular genetic alterations for the development of CRC and focused on the MAPK pathway, HIF-1 α and NOS2 genes.

    Alterations in the MAPK pathway have been found in several different cancer forms, including CRe. In the present study, we found somatic mutations in the MAPK pathway in 50% of the CRCs; 40% of the tumors carried mutations in the KRAS gene and 10% carried BRAF mutations. No genetic alterations were found in the ARAF or RAF-1 genes. B&4F gene mutations were present only in exon 15 and were associated with micro satellite instability. Three mutation types were identified; V599E, D593G and K600N, whereof the latter has not previously been described.

    The hypoxia inducible factor (HIF)-la protein is involved in the oxygen sensing mechanism and several tumor types show HIF-la overexpression due to hypoxia. At normoxia, HIF-la is degraded by interaction with the von Hippel-Lindau (VHL) tumor suppressor protein followed by an ubiquitin-proteasome dependent degradation mechanism, which prevents HIF-l a from nuclear translocation and transcription of downstream target genes. Fifteen percent of CRC patients and normal healthy population was found to carry the P582S polymorphism in the HIF-1 α gene, which previously has been associated to higher transactivating capacity. In the present study, the polymorphism was associated to ulcerative tumor development. In addition, loss of heterozygosity of the wild type P582 allele in heterozygotes may contribute to the development of ulcerative CRCs. However, the overall mechanism for ulcerative tumor development is still unclear.

    Nitric oxide (NO) is involved in several physiological processes, such as apoptosis, neurotransmission, angiogenesis and immune defence and is produced by three nitric oxide synthases; NOSl-3. In the present study, NOS2 upregulation was identified in CRCs compared to normal intestinal mucosa. Moreover, the contribution of NOS2 in CRC development was investigated in APCMin/+ and APCMin/+ NOS2-/- mice. The APCMin/+ NOS-/- mice developed a higher polyp frequency compared to APCMin/+ mice, indicating a protective role for the presence of NOS2 in intestinal cancer development. The elevated polyp formation in the APCMin/+ NOS-/- mice was independent of the expression of Notch-l and p21. We also investigated whether polymorphisms in the NOS2 promoter affected the onset of CRC, but no differences in allele or genotype frequencies were observed in normal healthy population compared to CRC patients.

  • 109.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dimberg, Jan
    Österström, Anna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Olsson, Anneli
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sirsjö, Allan
    Nitric oxide synthase 2 mRNA expression in relation to p53 and adenomatous polyposis coli mutations in primary colorectal adenocarcinomas2002In: Surgery, ISSN 0039-6060, E-ISSN 1532-7361, Vol. 131, no 4, p. 384-392Article in journal (Refereed)
    Abstract [en]

    Background. The inducible nitric (NO) synthase 2 (NOS2) is upregulated in breast, brain, colon, and gynecological tumors, which indicate that NO may have a role in tumorigenesis. However, little is known about the role and regulation of NOS2 in colorectal carcinomas. Recent in vitro experiments have implicated that NOS2 is downregulated by p53 accumulation. Virtual analysis of the NOS2 promoter showed putative TCF-4/Lef-1 response elements, which indicate a potential regulation of NOS2 expression by activation of the adenomatous polyposis coli (APC)/β-catenin pathway.

    Methods. NOS2 mRNA expression was investigated in 59 colorectal carcinomas by reverse transcriptase/real-time polymerase chain reaction and related to mutations in the p53, APC, and β-catenin genes. Presence of NOS2 protein was studied by Western blot, and the localization was studied by immunohistochemistry. Loss of heterozygosity was studied in the region of the NOS2 gene.

    Results. The NOS2 mRNA and protein expression were significantly higher in tumors than in control tissue. Immunohistochemistry revealed extensive NOS2 staining in the epithelial cells and, to a minor degree, in leukocytes. Increased NOS2 mRNA expression was found in Dukes' stages A and B compared with the C and D stages. No relationship was found between elevated NOS2 expression and loss of heterozygosity in the later stages according to Dukes' classification or mutations in the p53, APC, or β-catenin genes.

    Conclusions. Inactivating mutations in the p53 and APC pathways are not the main explanation for the increased NOS2 expression found in colorectal tumors.

  • 110.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Elander, Nils
    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.
    Nitric oxide synthase 2 (NOS2) promoter polymorphisms in colorectal cancer2005In: Cancer Letters, ISSN 0304-3835, E-ISSN 1872-7980, Vol. 225, no 1, p. 99-103Article in journal (Refereed)
    Abstract [en]

    Previously, increased expression of nitric oxide synthase 2 (NOS2) in colorectal cancer (CRC) has been identified. The NOS2 gene is transcriptionally regulated, which suggests that polymorphisms in the NOS2 promoter may have a role for CRC development and progression. The genotyping was performed with PCR/RFLP, single strand conformation analysis or MegaBACE genotyping of normal blood DNA from CRC patients and normal healthy controls. However, no significant association between NOS2 polymorphisms and CRC onset or clinical outcome was evident. In conclusion, these results, therefore, suggest that NOS2 promoter polymorphisms have a limited effect on the onset or progression of CRC.

  • 111.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fenech, Matthew
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Fredrikson, Mats
    Linköping University, Department of Molecular and Clinical Medicine, Occupational and Environmental Medicine. Linköping University, Faculty of Health Sciences.
    Dabrosin, Charlotta
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Association between ulcerative growth and hypoxia inducible factor-1α polymorphisms in colorectal cancer patients2006In: Molecular Carcinogenesis, ISSN 0899-1987, E-ISSN 1098-2744, Vol. 45, no 11, p. 833-840Article in journal (Refereed)
    Abstract [en]

    The hypoxia inducible factor-1α (HIF-1α) has been found to be involved in several different physiological mechanisms, such as blood-vessel formation, apoptosis, and erythropoiesis. HIF-1α is hydroxylated at normoxia and rapidly degraded via the von Hippel–Lindau (VHL)/ubiquitin-proteasome degradation system to prevent angiogenesis. In a previous study, the C1772T (P582S) and the G1790A (A588T) polymorphisms were identified in the human HIF-1α gene, which was shown to have a higher transactivating capability in vitro compared to the wild type allele. However, the role for these polymorphisms in vivo is still unclear. In the present investigation, we have therefore studied the role of the two polymorphic variants in the development of colorectal cancer (CRC) with PCR/RFLP (restriction fragment length polymorphism), single strand conformation analysis (SSCA), and immunohistochemistry (IHC). A significant higher-risk was identified between patients heterozygous for the C1772T polymorphism and the more severe ulcerative growth pattern compared to homozygous C1772C wild type tumors (RR = 5.2; 95% CI 1.26–21.6; P = 0.006). This was also verified on the allelic level (RR = 6.5; 95% CI 1.58–26.8; P = 0.001). In addition, patients carrying one or more polymorphic alleles in either the HIF-1α C1772T or the G1790A polymorphisms display significant higher risk for the development of ulcerative CRCs (RR = 4.17; 95% CI = 1.33–13.08; P = 0.004). These results suggest that the HIF-1α polymorpisms are an important factor for development of a subset of ulcerative intestinal tumors. Future screening of the polymorphic HIF-1α allele may therefore be of importance in the selection of treatment strategies of CRC.

  • 112.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Klintenäs, Maria
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Österström, Anna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dimberg, Jan
    Department of Natural Science and Biomedicine, University College of Health Sciences, Jönköping, Sweden.
    Monstein, Hans-Jürg
    Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Molecular Biological Techniques.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Mutation analysis of the BRAF, ARAF and RAF-1 genes in human colorectal adenocarcinomas2004In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 25, no 4, p. 527-533Article in journal (Refereed)
    Abstract [en]

    Colorectal cancer is a multi-step process characterized by a sequence of genetic alterations in cell growth regulatory genes, such as the adenomatous polyposis coli, KRAS, p53 and DCC genes. In the present study mutation analysis was performed with SSCA/direct sequencing of the hot-spot regions in exons 11 and 15 for the BRAF gene and exons 1–2 for the KRAS gene in 130 primary colorectal cancer tumors and correlated with clinico-pathological and mutational data. We also performed mutation analysis of the corresponding conserved regions in the ARAF and RAF-1 genes. Mutations in the BRAF and KRAS genes were found in 11.5 and 40% of the tumors, respectively. One germline exonic and nine germline intronic genetic variants were found in the ARAF and RAF-1 genes. All of the BRAF mutations were located in the kinase domain of the conserved region 3 in exon 15 of the BRAF gene. One novel somatic mutation was also identified in the BRAF gene. The majority of the BRAF mutations were found in colon compared with rectal tumors (P = 0.014). In agreement with others, a statistically significant correlation between BRAF mutations and microsatellite instability could be found. A negative correlation was also evident between mutations in the BRAF and KRAS genes, which supports earlier studies where somatic mutations in these genes are mutually exclusive. Collectively, our results provide support for the idea that activation of the MAP kinase pathway, especially via BRAF and KRAS mutations, is of critical importance for the development of colorectal cancer.

  • 113.
    Fransén, Karin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Sirsjö, A.
    Department of Caring Sciences, Örebro University, Örebro, SWEDEN.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Promotion of intestinal polyposis in nitric oxide synthase 2 (NOS2) deficient Min mice and expression of genes in the Notch-1 pathwayManuscript (preprint) (Other academic)
    Abstract [en]

    Nitric oxide synthase 2 (NOS2) expression has been found in several different tumor types, including colorectal cancers, but the role of NOS2 expression for cancer development is not fully understood. In the present study, we have investigated the role of NOS2 for intestinal polyp development in the APC Min/+ mouse and studied the mRNA expression by real time PCR of Notch-1 and p21 in normal murine small intestinal tissue and polyps from APC Min/+ NOS2+/+ and APC Min/+ NOS2-/- mice. A significant higher polyp frequency was found in mice with APC Min/+ NOS2-/- genotype compared to APC Min/+ NOS2+/+ mice. The expression of Notch-1 was significantly increased in polyps from the APC Min/+ NOS2+/+ mice compared to wild type small intestinal mucosa, but no difference was evident between the APC Min/+ NOS2+/+ and APC Min/+ NOS2-/- mice, which indicates that NOS2 expression does not affect the Notch-1 expression. No significant difference was found between the different mouse groups regarding the expression of p21. Collectively, NOS2 expression is a protective factor in intestinal polyposis, but its role in polyp development is still unclear.

  • 114.
    Fried, K.
    et al.
    Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
    Nosrat, C.
    Department of Neuroscience, Karolinska Institute, Stockholm, Sweden.
    Lillesaar, Christina
    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.
    Molecular signaling and pulpal nerve development2000In: Critical Reviews in Oral Biology and Medicine, ISSN 1045-4411, E-ISSN 1544-1113, Vol. 11, no 3, p. 318-332Article in journal (Refereed)
    Abstract [en]

    The purpose of this review is to discuss molecular factors influencing nerve growth to teeth. The establishment of a sensory pulpal innervation occurs concurrently with tooth development. Epithelial/mesenchymal interactions initiate the tooth primordium and change it into a complex organ. The initial events seem to be controlled by the epithelium, and subsequently, the mesenchyme acquires odontogenic properties. As yet, no single initiating epithelial or mesenchymal factor has been identified. Axons reach the jaws before tooth formation and form terminals near odontogenic sites. In some species, local axons have an initiating function in odontogenesis, but it is not known if this is also the case with mammals. In diphyodont mammals, the primary dentition is replaced by a permanent dentition, which involves a profound remodeling of terminal pulpal axons. The molecular signals underlying this remodeling remain unknown. Due to the senescent deterioration of the dentition, the target area of tooth nerves shrinks with age, and these nerves show marked pathological-like changes. Nerve growth factor and possibly also brain-derived neurotrophic factor seem to be important in the formation of a sensory pulpal innervation. Neurotrophin-3 and -4/5 are probably not involved. In addition, glial cell line-derived neurotrophic factor, but not neurturin, seems to be involved in the control of pulpal axon growth. A variety of other growth factors may also influence developing tooth nerves. Many major extracellular matrix molecules, which can influence growing axons, are present in developing teeth. It is likely that these molecules influence the growing pulpal axons.

  • 115.
    Fried, K.
    et al.
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden and Center for Oral Biology, Novum, Karolinska Institutet, Huddinge, Sweden.
    Risling, M.
    Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden.
    Tidcombe, H.
    Division of Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.
    Gassmann, M.
    Division of Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London, United Kingdom.
    Lillesaar, Christina
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Expression of ErbB3, ErbB4, and neuregulin-1 mRNA during tooth development2002In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 224, no 3, p. 356-360Article in journal (Refereed)
    Abstract [en]

    The receptor tyrosine kinases ErbB3 and ErbB4, which bind to various variants of neuregulin (NRG), play fundamental roles in neural development and in organs, which form through epithelial-mesenchymal interactions. Here, we demonstrate that NRG-1 and the receptors ErbB3 and ErbB4 are expressed locally during rodent tooth development. However, the mRNA expression patterns of ErbB3 and ErbB4 were distinctly different during odontogenesis. Examinations of teeth in genetically heart-rescued ErbB4-/- mice did not reveal any obvious deviation from the normal phenotype. The results suggest that ErbB3 and ErbB4 may participate in tooth morphogenesis. The specific interactions between NRG isoforms and ErbB receptors during this process remain to be determined.

  • 116.
    Fällman, Maria
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Complement receptor-mediated signal transduction in human neutrophils: A role for protein kinase C in the phagocytic process1992Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The neutrophil granulocyte comprises the first line of human defense against invading microorganisms. A versatile motile machinery enables the cell to migrate to the inflammatory site and there engulf and destroy the pathogens by phagocytosis. Engulfment is facilitated by opsonization of the microbes with C3b or C3bi complement fragments or with immunoglobulins, proteins which respectively bind to complement receptors (CRs) and Fe receptors on the neutrophil surface. The aim of the present thesis was to investigate the transmembrane signaling events involved in receptor-mediated engulfment.

    Experimentially induced inhibition of the phagocytic capacity of human neutrophils could be reversed by pretrcating the cells with protein kinase C (PKC)-activating agents such as PMA and a synthetic diglyceride. This indirectly suggests an important role for PKC in the process of engulfment. Presentation of opsonized yeast particles to neutrophils stimulated the phosphoinositide signaling pathway, resulting in an accumulation of Ins(1,4,5)P3 (IP3) and diglyceride (DG; the endogenous activator of PKC) with a time kinetic correlating that of the cellular uptake of the particles. However, in calcium-depleted neutrophils, formation of IP3 was totally abolished during phagocytosis of complement-opsonized yeast particles, thereby excluding this signal as a regulator of the engulfment process. DG, on the other hand, was still produced in these cells, suggesting a source other than phosphatidylinositols for the generation of this second messenger. Further studies revealed that a major part of CR-mediated DG formation originated from phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PC). The presence of DG suggested a subsequent activation of PKC, which was confirmed by the demonstration of CR-mediated phosphorylation of a well-known PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS). Moreover, the activity of PLD was shown to be regulated by PKC, which stimulated the production of its own activator by enhancing the activity of the lipase: PKC should thereby be able to maintain its own activity.Furthermore, we could also show that both CRI and CR3 can mediate PLO activation and that the degree of this activation is potentiated by PMA pretreatment and dependent on the fonn of ligand presentation. In conclusion, CR-mediated phagocytosis is associated with PLDmediated hydrolysis of PC and stimulation of PKC, the activity of the latter enzyme appears to be an important regulatory event in the engulfment process.

  • 117.
    Gao, X
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Islam, Quamrul
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    A gene on pig chromosome 14 suppresses cellular anchorage independence of the mouse cell line GM052672001In: Cytogenetics and Cell Genetics, ISSN 0301-0171, E-ISSN 1421-9816, Vol. 94, no 1-2, p. 62-66Article in journal (Refereed)
    Abstract [en]

    We have generated pig-mouse somatic cell hybrids by fusing normal pig fibroblasts with an anchorage independent mouse cell line GM05267. High quality G-banding analysis was applied to a set of 18 hybrid cell lines derived from 15 independent hybrids and chromosomes were identified. Cytogenetic analysis showed that all hybrids contained one or several pig chromosomes with normal morphology devoid of any structural changes. Out of 18 hybrids tested for colony formation in soft agar, 15 were suppressed for anchorage independence while the remaining three were not suppressed. Correlation of the cellular phenotype with the pig chromosome content of the hybrids suggests that the suppressor function for anchorage independence is located on pig chromosome (SSC) 14. We have previously shown that a suppressor gene for anchorage independence (SAI1) is located on rat chromosome (RNO) 5 and another suppressor gene for the same phenotype is located on human chromosome (HSA) 9. Given the genetic homology of both RNO5 and HSA9 with two pig chromosomes including SSC14, the third suppressor gene we have mapped on SSC14 may well be a functional homologue of the previously identified rat and human genes.

  • 118. Geirsson, Gudmundur
    et al.
    Lindström, Sivert
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Fall, Magnus
    The bladder cooling reflex and the use of cooling as stimulus to the lower urinary tract.1999In: Journal of Urology, ISSN 0022-5347, E-ISSN 1527-3792, Vol. 162, p. 1890-1896Article in journal (Refereed)
  • 119.
    Gentile, Massimiliano
    et al.
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Bergman Jungeström, Malin
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    Olsen, K. E.
    Linköping University, Department of Neuroscience and Locomotion, Pathology. Linköping University, Department of Molecular and Clinical Medicine, Forensic Medicine. Linköping University, Faculty of Health Sciences.
    Söderkvist, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wingren, Sten
    Linköping University, Department of Biomedicine and Surgery, Oncology. Linköping University, Faculty of Health Sciences.
    p53 and survival in early onset breast cancer: analysis of gene mutations, loss of heterozygosity and protein accumulation1999In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 35, no 8, p. 1202-1207Article in journal (Refereed)
    Abstract [en]

    The p53 protein has proven to be central in tumorigenesis by its cell cycle regulatory properties and both gene mutations and protein accumulation have been associated with poor prognosis in breast cancer. The present study was undertaken to investigate the prognostic significance of gene mutations, p53 protein accumulation and of loss of heterozygosity (LOH) at the TP53 locus in young (age <37 years) breast cancer patients. In total, gene mutations were found in 21 of the 123 patients (17%), LOH in 20 of the 47 informative cases (43%) and protein accumulation in 47 of the 102 available cases (46%). Log rank analysis revealed no significant association between survival and TP53 mutations (in general), p53 protein accumulation or LOH. However, missense mutations localised to the zinc binding domain were significantly (P=0.0007) associated with poorer prognosis. As indicated in this as well as other studies, p53 protein accumulation is frequently found in young breast cancer patients, but this protein overexpression appears to be of minor significance for survival. Nevertheless, the present report also suggests that specific mutations contribute substantially to tumour aggressiveness.

  • 120. Graham, J
    et al.
    Kockum, I
    Sanjeevi, CB
    Landin-Olsson, M
    Nyström, L
    Sundkvist, G
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Blohmé, G
    Lithner, F
    Littorin, B
    Scherstén, B
    Wibell, L
    Östman, J
    Lernmark, Å
    Breslow, N
    Dahlquist, G
    Negative asociation between type 1 diabetes and HLA DQB1*0602-DQA1*0102 is attenuated with age at onset.1999In: European journal of immunogenetics, ISSN 0960-7420, E-ISSN 1365-2370, Vol. 26, p. 117-127Article in journal (Refereed)
  • 121.
    Granseth, B
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ahlstrand, Erik
    Lindstrom, S
    Effects of extracellullar calcium ions on paired pulse facilitation and depression in the dorsal lateral geniculate nucleus in vitro.2000Conference paper (Other academic)
  • 122.
    Granseth, Björn
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dynamic properties of corticogeniculate excitatory transmission in the rat dorsal lateral geniculate nucleus in vitro2004In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 556, no 1, p. 135-146Article in journal (Refereed)
    Abstract [en]

    The feedback excitation from the primary visual cortex to principal cells in the dorsal lateral geniculate nucleus (dLGN) is markedly enhanced with firing frequency. This property presumably reflects the ample short-term plasticity at the corticogeniculate synapse. The present study aims to explore corticogeniculate excitatory postsynaptic currents (EPSCs) evoked by brief trains of stimulation with whole-cell patch-clamp recordings in dLGN slices from DA-HAN rats. The EPSCs rapidly increased in amplitude with the first two or three impulses followed by a more gradual growth. A double exponential function with time constants 39 and 450 ms empirically described the growth for 5–25Hz trains. For lower train frequencies (down to 1Hz) a third component with time constant 4.8 s had to be included. The different time constants are suggested to represent fast and slow components of facilitation and augmentation. The time constant of the fast component changed with the extracellular calcium ion concentration as expected for a facilitation mechanism involving an endogenous calcium buffer that is more efficiently saturated with larger calcium influx. Concerning the function of the corticogeniculate feedback pathway, the different components of short-term plasticity interacted to increase EPSC amplitudes on a linear scale to firing frequency in the physiological range. This property makes the corticogeniculate synapse well suited to function as a neuronal amplifier that enhances the thalamic transfer of visual information to the cortex.

  • 123.
    Granseth, Björn
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    The corticogeniculate synapse: a neuronal amplifier?2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Consciousness is a nervous process that handles only a limited amount of information. Therefore the nervous system needs to select the most relevant input for aware processing. For the visual system, it has been suggested that recurrent excitation from the cortex to neurones in the lateral geniculate nucleus provides a "spotlight of attention", that selectively enhances the relay of information to the cortex. Such feedback excitation could be supplied by corticogeniculate neurones in layer 6 of the primary visual cortex. The corticogeniculate synaptic strength increases with neuronal firing frequency. From this property it can be hypothesised that the feedback excitation would function as a variable neuronal amplifier for boosting the information transfer in the attentive state. The general aim of this thesis was to study the synaptic mechanisms that make the corticogeniculate synapse frequency sensitive and evaluate this property in relation to the proposed neuronal amplifier function.

    Experiments were performed with whole-cell patch-clamp recordings from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Ex citatory postsynaptic currents (EPSCs) evoked by stimulation of corticogeniculate axons consistently displayed paired pulse facilitation. The ratio EPSC2 I EPSC1 was 3.7 ± 1.6 (mean ± standard deviation) for two pulses separated 40 ms. The paired pulse facilitation comprised a fast and slow component, evident from its double exponential decay. EPSCs evoked in the same cells by stimulating axons from the retina displayed paired pulse depression. The two types of EPSCs differed in their response to alterations in the extracellular calcium ion concentration ([Ca2+]o). The paired pulse depression at retinogeniculate synapses was attenuated by decreasing [Ca2+]o, apparently from lowering the level of transmitter release. At the corticogeniculate synapse, paired pulse facilitation was optimal at physiological [Ca2+]o. The facilitation was presynaptic in origin since the facilitated EPSC2 quantal size (q = - 5.2 ± 0.8 pA) was essentially the same as for EPSC1 (q = - 4.9 ± 0.9 pA). Each corticogeniculate axon terminated with 1 - 2 functional synapses (nsyn) per principal cell and the basal transmitter release probability was low (psyn = 0.09 ± 0.04) but increased with facilitation (psyn = 0.25 ± 0.10).

    When short trains of pulses were used for stimulation of corticogeniculate axons, the EPSCs rapidly increased in amplitude with the first 2 - 3 stimuli followed by a more gradual growth. A double exponential function, likely to represent the fast and slow components of facilitation could describe the EPSC build-up in amplitude. The time constant of fast facilitation was dependent on [Ca2+]o , presumably representing Ca2+ binding to a saturable intraterminal Ca2+ buffer. When pulse trains were repeated at 1 - 10 s intervals, EPSC1 in each train was progressively enhanced by augmentation, leaving late EPSCs unaffected. When [Ca2+]o was altered, augmented EPSCs changed in proportion to the basal EPSC amplitude, i.e. EPSC1:n / EPSC1,1 remained the same. The results indicate that augmentation is determined by a Ca2+ residue in the presynaptic terminal after repetitive spike firing, competing with the mechanism of the fast component of facilitation.

    The two components of facilitation and augmentation at the corticogeniculate synapse define the function of the suggested neuronal amplifier. The low basal synaptic strength ascertains that single random spikes will be virtually ineffective at the target cell, which protects the ex citatory feedback system from self-generated cyclic activity. Since the different forms of synaptic enhancement are presynaptic, the neuronal amplifier will be strictly stimulus specific in increasing synaptic strength. Furthermore, the different components seem to interact to increase EPSC amplitudes on a linear scale to firing frequency, that will increase the dynamic range of neuronal firing without distorting the basic characteristics of thalamic relay. Fast facilitation would account for most of the gain of the neuronal amplifier, while augmentation primarily reduces the time required to reach an effective level of synaptic strength. Thus it might serve to preserve the gain of the neuronal amplifier during attentive visual exploration, when the gaze may return repeatedly to the same fixation point.

  • 124.
    Granseth, Björn
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ahlstrand, Erik
    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.
    Paired pulse facilitation of corticogeniculate EPSCs in the dorsal lateral geniculate nucleus of the rat investigated in vitro2002In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 544, no 2, p. 477-486Article in journal (Refereed)
    Abstract [en]

    To investigate paired pulse facilitation of corticogeniculate EPSCs, whole-cell patch-clamp recordings were made from principal cells in the rat dorsal lateral geniculate nucleus (dLGN) in vitro. Thalamic slices, oriented so that both corticogeniculate and retinogeniculate axons could be stimulated, were cut from young (16- to 37-day-old) DA-HAN rats. Corticogeniculate EPSCs displayed pronounced paired pulse facilitation at stimulus intervals up to 400 ms. The facilitation had a fast and a slow component of decay with time constants of 12 ± 7 and 164 ± 47 ms (means ± s.d.), respectively. Maximum paired pulse ratio (EPSC2 × EPSC1−1) was 3.7 ± 1.1 at the 20-30 ms interval. Similar to other systems, the facilitation was presynaptic. Retinogeniculate EPSCs recorded in the same dLGN cells displayed paired pulse depression at intervals up to at least 700 ms. The two types of EPSCs differed in their calcium response curves. At normal [Ca2+]o, the corticogeniculate synapse functioned over the early rising part of a Hill function, while the retinogeniculate synapse operated over the middle and upper parts of the curve. The paired pulse ratio of corticogeniculate EPSCs was maximal at physiological [Ca2+]o. The facilitation is proposed to have an important role in the function of the corticogeniculate circuit as a neuronal amplifier.

  • 125.
    Granseth, Björn
    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.
    Augmentation of corticogeniculate EPSCs in principal cells of the dorsal lateral geniculate nucleus of the rat investigated in vitro2004In: Journal of Physiology, ISSN 0022-3751, E-ISSN 1469-7793, Vol. 556, no 1, p. 147-157Article in journal (Refereed)
    Abstract [en]

    Augmentation is a component of short-term synaptic plasticity with a gradual onset and duration in seconds. To investigate this component at the corticogeniculate synapse, whole cell patch-clamp recordings were obtained from principal cells in a slice preparation of the rat dorsal lateral geniculate nucleus. Trains with 10 stimuli at 25 Hz evoked excitatory postsynaptic currents (EPSCs) that grew in amplitude, primarily from facilitation. Such trains also induced augmentation that decayed exponentially with a time constant τ= 4.6 ± 2.6 s (mean ± standard deviation). When the trains were repeated at 1–10 s intervals, augmentation markedly increased the size of the first EPSCs, leaving late EPSCs unaffected. The magnitude of augmentation was dependent on the number of pulses, pulse rate and intervals between trains. Augmented EPSCs changed proportionally to basal EPSC amplitudes following alterations in extracellular calcium ion concentration. The results indicate that augmentation is determined by residual calcium remaining in the presynaptic terminal after repetitive spikes, competing with fast facilitation. We propose that augmentation serves to maintain a high synaptic strength in the corticogeniculate positive feedback system during attentive visual exploration.

  • 126.
    Granseth, Björn
    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.
    Unitary EPSCs of corticogeniculate fibers in the rat dorsal lateral geniculate nucleus in vitro2003In: Journal of Neurophysiology, ISSN 0022-3077, E-ISSN 1522-1598, Vol. 89, no 6, p. 2952-2960Article in journal (Refereed)
    Abstract [en]

    To investigate unitary corticogeniculate excitatory postsynaptic currents (EPSCs), whole cell patch-clamp recordings were obtained from 20 principal cells in slices of the dorsal lateral geniculate nucleus (dLGN) of DA-HAN rats. EPSCs, evoked by electrical stimulation of corticogeniculate axons, had size distributions with one or more quantal peaks. Gaussian curves fitted to such distributions gave a mean quantal size (q) of -5.0 ± 0.7 (SD) pA for the EPSCs. Paired-pulse ratio (EPSC2/EPSC1) was 3.3 ± 0.9 for stimuli separated by 40 ms. The mean quantal size was similar for facilitated EPSCs (-5.2 ± 0.8 pA), implying an increase in mean quantal content (m). Most corticogeniculate axons were capable of releasing only one or two quanta onto individual principal cells. Mean resting release probability (p) was low, 0.09 ± 0.04. Binomial models, with the same n but increased p, could account for both the basal and facilitated EPSC size distributions in 6/8 cells. It is suggested that the low resting efficacy of corticogeniculate synapses serves to stabilize this excitatory feedback system. The pronounced facilitation in conjunction with large convergence from many corticogeniculate cells would provide a transient, potent excitation of dLGN cells, compliant with the idea of a visually driven neuronal amplifier.

  • 127.
    Grönroos, Eva
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Leukotriene D4-induced signal transduction in human epithelial cells1996Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Although the structures of the cysteinyl-leukotrienes have been known for almost twenty years, and their biological effects for almost sixty years, very little is known about the signaling propenies of these compounds. The aim of the present research was to further elucidate the components that are involved in the signaling mechanisms of leukotriene D4 (L TD4) in a human epithelial cell line.

    Stimulation with LTD4 was found to induce the mobilization of intracellular calcium as well as the influx of calcium through the plasma membrane. Although the LTD4 receptor probably belongs to the seven-transmembrane-spanning G-protein-coupled receptors, phospholipase Cyt is tyrosine phosphorylated and translocated to the plasma membrane upon stimulation with L1D4. The unknown, genistein-insensitive kinase responsible for this effect is directly or indirectly activated by a monomericG-protein that probably belongs to the Rho fantily. We were unable to see any direct association between a Rho protein and phospholipase Cy1, instead we found that the monomeric G-protein Rapl associates with phospholipase Cyt in unstimulated cells.

    After stimulation the content of Rapl increases in the plasma membrane,simultaneously as it dissociates from phospholipase Cy1 in a manner dependent on protein kinase A. The L1D4-induced calcium influx is mediated through a pertussistoxin- sensitive 0-protein. Engagement of the L TD4 receptor also induces a rapid increase in cAMP and the activation of a genistein-sensitive tyrosine kinase. Both of these events are necessary for the LTD4-induced calcium influx. The increase in cAMP probably precedes the activation of the tyrosine kinase, since preincubation with Rp-cAMPS, which inhibits protein kinase A, interferes with the L 1D4-induced tyrosine phosphorylation. The identities of the kinases involved in the calcium mobilization and influx are still unresolved.

    Taken together, our findings show that L TD4 stimulation of the intestinal epithelial cell line INT 407 results in the activation of at least three different G-proteins and two kinases of importance for the regulation of cytosolic calcium levels in these cells.

  • 128.
    Guidez, F
    et al.
    Chester Beatty Laboratories, London, UK.
    Ivins, S
    Chester Beatty Laboratories, London, UK.
    Zhu, J
    Chester Beatty Laboratories, London, UK.
    Söderström, Mats
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Waxman, S
    Mount Sinai School of Medicine, New York, USA.
    Zelent, A
    Chester Beatty Laboratories, London, UK.
    Reduced retinoic acid-sensitivities of nuclear receptor corepressor binding to PML- and PLZF-RARalpha underlie molecular pathogenesis and treatment of acute promyelocytic leukemia1998In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 91, no 8, p. 2634-2642Article in journal (Refereed)
    Abstract [en]

    Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARalpha fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RARalpha fusion protein. Both PML- and PLZF-RARalpha possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RARalpha protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RARalpha, it had a very little effect on its association with the PLZF-RARalpha fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RARalpha was mediated by the N-terminal PLZF moiety of the chimera. It appears that N-CoR/histone deacetylase corepressor complex interacts directly in an ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF protein and is required, at least in part, for its function as a transcriptional repressor. As the above-noted results predict, histone deacetylase inhibitors antagonize oncogenic activities of the PML-RARalpha fusion protein and partially relieve transcriptional repression by PLZF as well as inhibitory effect of PLZF-RARalpha on ATRA response. Taken together, our results demonstrate involvement of nuclear receptor corepressor/histone deacetylase complex in the molecular pathogenesis of APL and provide an explanation for differential sensitivities of PML- and PLZF-RARalpha-associated leukemias to ATRA.

  • 129.
    Gunnarsson, Peter
    et al.
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    Levander, Louise
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Påhlsson, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Grenegård, Magnus
    Linköping University, Department of Medicine and Health Sciences, Pharmacology . Linköping University, Faculty of Health Sciences.
    The acute-phase protein alpha 1-acid glycoprotein (AGP) induces rises in cytosolic Ca2+ in neutrophil granulocytes via sialic acid binding immunoglobulin-like lectins (siglecs)2007In: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, ISSN 1530-6860, Vol. 21, no 14, p. 4059-4069Article in journal (Refereed)
    Abstract [en]

    We studied whether the acute-phase protein alpha1-acid glycoprotein (AGP) induces rises in [Ca2+]i in neutrophils and sought to identify the corresponding AGP receptor (or receptors). We found that AGP elicited a minimal rise in [Ca2+]i in Fura-2-loaded neutrophils, and this response was markedly enhanced by pretreatment with anti-L-selectin antibodies. (The EC50 value of the AGP-induced Ca2+ response was 9 microg/ml.) Activation of phospholipase-C, Src tyrosine kinases, and PI3 kinases proved to be essential for the AGP-mediated increase in [Ca2+]i, whereas the p38 MAPK and SYK signaling pathways were not involved. Furthermore, antibodies against sialic acid binding, immunoglobulin-like lectin 5 (Siglec-5) and oligosaccharide 3'-sialyl-lactose both antagonized the AGP-induced response and caused an immediate increase in [Ca2+]i in anti-L-selectin-treated neutrophils, which indicates a signaling capacity of Siglec-5. We used modified forms of AGP (treated with mild periodate or neuraminidase) to establish the importance of sialic acid residues. The modified forms of AGP caused a much smaller rise in [Ca2+]i than did unaltered AGP. Affinity chromatography confirmed that unchanged AGP, but not neuraminidase-treated AGP, bound to Siglec-5. Our report provides the first evidence for a signaling capacity by AGP through a defined receptor. Pre-engagement of L-selectin significantly enhanced this signaling capacity.

  • 130.
    Gustafsson, T
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Andersson, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Different inhibitory actions of IGFBP-1, -2 and-4 on IGF-1 effects in vascular smooth muscle cells.1999In: Journal of Endocrinology, ISSN 0022-0795, E-ISSN 1479-6805, Vol. 161, p. 245-253Article in journal (Refereed)
  • 131.
    Gustafsson, T
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Andersson, Peter
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Chen, Y
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Magnusson, JO
    Magnusson, JO
    Arnqvist, Hans
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Interaction of angiotensin II and the insulin-like growth factor system in vascular smooth muscle cells.1999In: American journal of physiology, ISSN 0002-9513, Vol. 277, p. 499-507Article in journal (Refereed)
  • 132.
    Gustafsson, Thomas
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, The Institute of Technology.
    Regulation and actions of insulin-like growth factor binding proteins in vascular smooth muscle cells1999Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Insulin-like growth factor-I (IGF-I) has multiple actions on vascular smooth muscle cell (VSMCs) function. In the circulation and in extracellular fluids, IGF-I is complexed to high affinity IGF-binding proteins (IGFBPs), which modulate IGF-I bioactivity. The present study focused on expression and regulation of IGFBPs in VSMCs. and how they modulate IGF-1 effects in these cells.

    Rar VSMCs expressed mRNAs for IGFBP-2, -4 and -6, while human VSMCs expressed mRNAs for IGFBP-2, -3, -4, -5 and -6 as determined by solution hybridization. IGF-1 (10-8 M) increased and angiotensin TI (AII, 10-6 M) decreased IGFBP-2 and IGFBP-4 mRNA in rat VSMCs. Furthermore, AII and All and 1GF-I in combination increased IGF-I receptor mRNA and All decreased IGF-1 mRNA. We detected endogenous 1GFBP-2 and IGFBP-4 by Western immuno blot in concentrated conditioned medium of rat VSMCs. IGF-I did not affect steadystate levels of IGFBP-2 and -4, but increased the amount of fragments of these IGFBPs. Exogenously added IGFBP-2 and -4 were minimally degraded in serum free conditioned medium for up to 72h in the presence of VSMCs, but immunoreactive bands of the intact IGFBPs disappeared in the presence of IGF-I. We detected IGFBP-2, -4, -5 and -6 in concentrated conditioned medium of human VSMCs.

    IGFBP-1 and IGFBP-4 inhibited 1GF-1 (1 nM) induced DNA synthesis in rat VSMCs with IC50 values of 1.6 and 6.2 nM respectively, and they also inhibited IGF-I induced protein synthesis. IGFBP-2, if preincubated with IGF-I, also acted inhibitory on IGF-I action. IGFBP-1, -3 and- 4 (1.4-2.0 nM) inhibited IGF-I (I nM) induced DNA synthesis in human VSMCs, while IGFBP-2, -5 and -6 had no effects in these concentrations.

    IGF-I and AII had additive effects on DNA- and protein synthesis in rat VSMCs and these effects occurred at doses, at which the peptides added alone initiated DNA synthesis. Losartan blocked the synergistic effects and IGFBP-1, which inhibited IGF-I action, was not able to inhibit the action of AII.

    In conclusion, rat and human VSMCs express IGFBP-2, -4 and -6, but not IGFBP-1. The results on IGFBP-3 and IGFBP-5 are inconsistent. IGFBP-1, -3 and -4 act at physiological concentrations inhibitory on IGF-I stimulated effects. IGFBP-2 and IGFBP-4 are metabolized by proteases from rat and human VSMCs and the degradation of these IGFBPs is enhanced by IGF-I. There is an interaction of AII and the IGF-system in VSMCs and IGF-I and All have additive effects on DNA- and protein synthesis in these cells.

  • 133.
    Gustavsson, Johanna
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Insulin control of glucose transport in caveolae microdomains of the plasma membrane1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Caveolae are invaginated, dynamic microdomains in the plasma membrane and believed to be involved in receptor-mediated uptake of small molecules (potocytosis) and in signal transduction. A phosphatidylinositol glycan, a precursor of potential insulin second messengers, has been found to be enriched in the caveolae-fraction of adipocyte plasma membranes (Parpal et al., 1995, J Cell Biol 131:125-135). We now demonstrate that the insulin receptor is localized to caveolae microdomains. This was investigated in i) 3T3-Ll adipocyte plasma membranes by a morphological method (double immunofluorescence labeling and contbcal microscopy) and in ii) caveolae isolated by a biochemical, detergent-free method. The insulin receptor was enriched in caveolae and, in response to insulin, phosphorylated on tyrosine which indicated that the insulin receptor was active.

    Insulin stimulates the translocation of glUcose transporter protcins from intracellular stores to the plasma membrane which leads to an increased glucose uptake. Long-chain 1 ,2-diacylgiycerol, one of two potential second messengers for insulin, has been found to stimulate glucose uptake in rat adipocytes (Stn\1fors, 1988, Nature, 335:554-556). Here, we report that long-chain 1,2-diacylglycerol, emulsified in taurodeoxycholate, stimulates the translocation of GLUT4 to the plasma membrane. Moreover, physiological long-chain 1,2~diacylglycerols are taken up by different cell types in amounts sufficient to have biological eftects, equally well in the absence or presence of taurodeoxycholate.

    We also report that a rapid translocation of GLUT4 to the plasma membrane was followed by a slower transition of GLUT4 into caveolae. Accumulation of GLUT4 in caveolae coincided with the insulin-stimulated increase in glucose uptake. This offers a mechanistic explanation for the observed discrepancy between the appearance of GLUT4 in the plasma membrane and the delayed increase in glucose uptake.

    Non-hydrolyzable GTP-analogs stimulate the translocation of GLUT4 and increase glucose uptake in permeabilized cells. The small GTP·binding protein RaM is suggested to be involved in these processes since Rab4 has been localized to GLUT4-containing vesicles and is redistributed in response to insulin. We found that Rab4 is enriched in caveolae and that the amount of Rab4 increased in caveolae, in the same extent a<> GLUT4 did, in re!.J)Onse to insulin.

    Caveolae are characterized by high levels of sphingolipids and cholesterol. Depletion of cholesterol, which disrupts the integrity of caveolae, abolished insulin-stimulated glucose uptake reversibly. Insulin's control of protein pho.<:phorylation was also abolished while j3-adrenergic signaling was unaffected.

    The results suggest that caveolae are crucial Jor insulin-signuling in adipocytes and a disruption of these structures may have consequences for the development of insulin re.~istance and diabetes mellitus.

  • 134.
    Gustavsson, Johanna
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Parpal, Santiago
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Karlsson, Margareta
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Ramsing, Cecilia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Thorn, Hans
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Borg, Marie
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Lindroth, Margaretha
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Holmgren Peterson, Kajsa
    Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. 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.
    Strålfors, Peter
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Localization of the insulin receptor in caveolae of adipocyte plasma membrane1999In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 13, no 14, p. 1961-1971Article in journal (Refereed)
    Abstract [en]

    The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with β-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. β-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.

  • 135.
    Hallbeck, Martin
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Dynorphin mRNA-expressing neurons in the rat paraventricular hypothalamic nucleus project to the spinal cord2000In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 285, no 3, p. 161-164Article in journal (Refereed)
    Abstract [en]

    The opioid peptide dynorphin is important for the regulation of neuronal activity in the spinal cord. Because dynorphin is produced by neurons throughout the neuraxis, there are many putative sources for spinal dynorphin fibers, in addition to those originating from spinal cord neurons. Using a sensitive double-labeling technique combining in situ hybridization and tract tracing, the present study demonstrates that the paraventricular hypothalamic nucleus (PVH) of adult naı̈ve male Sprague–Dawley rats contains large numbers of dynorphin mRNA-producing cells with projections to the spinal cord. Thus, more than 40% of the spinally projecting neurons in PVH were found to express dynorphin mRNA. This novel finding suggests that the PVH is a major source of spinal dynorphin that may be of importance for the processing of pain and visceral information.

  • 136.
    Hallbeck, Martin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Effect of Stimulation of the Paraventricular Hypothalamic Nucleus on Noxious-Evoked Fos-immunoreactlvity In the Rat lumbar Spinal CordManuscript (preprint) (Other academic)
    Abstract [en]

    The paraventricular nucleus of the hypothalamus (PVH) provides a prominent descending projection to the superficial dorsal horn, and contains a number of neuropeptides that are know to influence nociceptive processing. In the present study, we injected formalin subcutaneously into the hind paws of unanesthetized rats and studied the noxious-evoked Fos protein expression in the dorsal horn following simultaneous unilateral injection of the glutamate receptor agonist kainic acid into the PVH. Although some cases displayed less Fos-inununoreactivity in the lumbar spinal cord on the side ipsilateral to the PVH activation than on the contralateral side, others displayed no side differences, and one case showed more labeling in the ipsilateral dorsal horn than on the contralateral side, Because different parts of the PVH were activated in the different experiments, the present observations suggest that the different peptide expressing populations of spinal cordprojecting neurons in PVH may have different, and perhaps opposing functions in the spinal dorsal horn.

  • 137.
    Hallbeck, Martin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Spinal cord-projecting vasopressinergic neurons in the rat paraventricular hypothalamus1999In: Journal of Comparative Neurology, ISSN 0021-9967, E-ISSN 1096-9861, Vol. 411, no 2, p. 201-211Article in journal (Refereed)
    Abstract [en]

    The paraventricular hypothalamic nucleus (PVH) is a key structure for the maintenance of homeostasis. Homeostatic regulation includes modulation of signaling in the spinal cord. This may be exerted by neurons in the PVH with spinal projections. However, the PVH is not a homogeneous structure, but consists of anatomically and functionally distinct subdivisions. In this study, we have analyzed the distribution of spinal cord-projecting PVH neurons that express vasopressin, an important neuropeptide in autonomic regulation. Vasopressinergic neurons were identified with a radiolabeled riboprobe complementary to vasopressin mRNA combined with immunohistochemical labeling of retrogradely transported cholera toxin subunit b in spinally projecting neurons. More than 40% of the spinally projecting neurons in the PVH of naive Sprague-Dawley rats were found to express vasopressin mRNA. The lateral parvocellular subdivision and the ventral part of the medial parvocellular subdivision contained the densest distribution of spinal cord-projecting vasopressin mRNA-expressing neurons. The magnocellular subdivisions displayed large numbers of vasopressin mRNA-expressing neurons, but very few of those projected to the spinal cord. The dorsal parvocellular subdivision contained a large number of spinally projecting neurons, but very few of those expressed vasopressin mRNA. These findings show that the PVH gives rise to a major vasopressinergic projection to the spinal cord and that the spinal cord-projecting vasopressinergic neurons are parceled into anatomically distinct cell groups. This provides an anatomical basis for a selective activation of functionally different groups in the PVH as part of a behaviorally adaptive response, including modulation of autonomic activity and pain processing at the spinal level.

  • 138.
    Hallbeck, Martin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hermanson, Ola
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Preprovasopressin mRNA is not present in dorsal root ganglia of the rat1996In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 209, no 10, p. 125-128Article in journal (Refereed)
    Abstract [en]

    Immunohistochemical studies on colchic ine-treated rats have suggested that more than half of the neurons in dorsal root ganglia (DRG) contain vasopressin. Thus, vasopressin would be the most commonly found peptide in DRG neurons. In the present study we have reexamined the presence of vasopressin in DRG neurons, using a sensitive in situ hybridization method employing long riboprobes that will detect very small amounts of mRNA. The C3, C6, T2, T12, L2 and L5 DRG were studied. None of these ganglia contained any preprovasopressin mRNA. Yet, dense labeling for preprovasopressin mRNA was seen on simultaneously processed hypothalamic sections and a heavy preprotachykinin mRNA expression was seen in adjacent DRG sections. These findings demonstrate that vasopressin is not produced in DRG in normal rats.

  • 139.
    Hallbeck, Martin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Hermansson, Ola
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Distribution of preprovasopressin mRNA in the rat central nervous system1999In: Journal of Comparative Neurology, ISSN 0021-9967, E-ISSN 1096-9861, Vol. 411, no 2, p. 181-200Article in journal (Refereed)
    Abstract [en]

    Vasopressin released in the central nervous system has been shown to be involved both in homeostatic mechanisms (e.g., water balance, thermoregulation, cardiovascular regulation, metabolism, and antinociception) and in higher brain functions (e.g., social recognition and communication, and learning and memory). Many nuclear groups have been proposed to synthesize vasopressin, but available data are conflicting. We have used a sensitive in situ hybridization technique to identify the distribution of the neurons that may be the origin of the vasopressin in the central nervous system of the male Sprague-Dawley rat. Vasopressin mRNA-expressing neurons were most abundant in the hypothalamus (e.g., the paraventricular, supraoptic, and suprachiasmatic nuclei) but were also seen in the medial amygdaloid nucleus, the bed nucleus of stria terminalis, and the nucleus of the horizontal diagonal band. Previously unreported vasopressinergic neurons were seen in the entorhinal and piriform cortices, the ventral lateral portion of the parabrachial nucleus, the pedunculopontine nucleus, and the rostral part of the ventral periaqueductal gray matter and the adjacent portion of the mesencephalic reticular nucleus. Vasopressin mRNA expression suggestive of neuronal labeling was seen in the pyramidal layer of the CA1–3 fields and the dentate gyrus of the hippocampus. In addition, vasopressin mRNA expression, probably representing axonal mRNA, was detected over the hypothalamopituitary tract. No or insignificant preprovasopressin mRNA expression was present in the cerebellum, locus coeruleus, subcoeruleus, or the spinal cord. These findings provide novel information on the distribution of vasopressin neurons that are important for our understanding of how vasopressin acts in the brain.

  • 140.
    Hallbeck, Martin
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Larhammar, Dan
    Department of Neuroscience, Unit of Pharmacology, Uppsala University, Sweden.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Neuropeptide expression in rat paraventricular hypothalamic neurons that project to the spinal cord2001In: Journal of Comparative Neurology, ISSN 0021-9967, E-ISSN 1096-9861, Vol. 433, no 2, p. 222-238Article in journal (Refereed)
    Abstract [en]

    The paraventricular hypothalamic nucleus (PVH) exerts many of its regulatory functions through projections to spinal cord neurons that control autonomic and sensory functions. By using in situ hybridization histochemistry in combination with retrograde tract tracing, we analyzed the peptide expression among neurons in the rat PVH that send axons to the spinal cord. Projection neurons were labeled by immunohistochemical detection of retrogradely transported cholera toxin subunit B, and radiolabeled long riboprobes were used to identify neurons containing dynorphin, enkephalin, or oxytocin mRNA. Of the spinally projecting neurons in the PVH, approximately 40% expressed dynorphin mRNA, 40% expressed oxytocin mRNA, and 20% expressed enkephalin mRNA. Taken together with our previous findings on the distribution of vasopressin-expressing neurons in the PVH (Hallbeck and Blomqvist [1999] J. Comp. Neurol. 411:201–211), the results demonstrated that the different PVH subdivisions display distinct peptide expression patterns among the spinal cord–projecting neurons. Thus, the lateral parvocellular subdivision contained large numbers of spinal cord–projecting neurons that express any of the four investigated peptides, whereas the ventral part of the medial parvocellular subdivision displayed a strong preponderance for dynorphin- and vasopressin-expressing cells. The dorsal parvocellular subdivision almost exclusively contained dynorphin- and oxytocin-expressing spinal cord–projecting neurons. This parcellation of the peptide-expressing neurons suggested a functional diversity among the spinal cord–projecting subdivisions of the PVH that provide an anatomic basis for its various and distinct influences on autonomic and sensory processing at the spinal level.

  • 141.
    Hamlin, Lina
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Medicine and Health Sciences.
    Mackerlova, Ludmila
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Medicine and Health Sciences.
    Blomqvist, Anders
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Medicine and Health Sciences.
    Ericson, Ann-Charlott
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Medicine and Health Sciences.
    AMPA-selective glutamate receptor subunits and their relation to glutamate-and GABA-like immunoreactive terminals in the nucleus submedius of the rat1996In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 217, no 2-3, p. 149-52Article in journal (Refereed)
    Abstract [en]

    Glutamate plays an important role in supraspinal nociceptive systems. Thus, glutamate is present in the nucleus submedius of the medial thalamus, a major relay for nociceptive information. In this study, immunoreactivity for the four subunits (GluR1-4) of alpha-amino-3-hydroxy-5-methyl-4-isoxasoleproprionate (AMPA) receptors was examined by a preembedding immunohistochemical method in order to evaluate the presence of this glutamate receptor subtype in the nucleus submedius. Combining the preembedding method with a postembedding immunogold technique, we found that AMPA receptor-like immunoreactivity was present postsynaptically to glutamatergic terminals but not to terminals containing gamma-aminobutyric acid (GABA). These findings suggest a role for AMPA receptors in excitatory synaptic transmission in the nucleus submedius of the rat thalamus.

  • 142.
    Hammarström, Sven
    et al.
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Trinks, Cecilia
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Wigren, Jane
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Surapureddi, Sailesh
    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.
    Glass, Christopher K
    University of California, San Diego, Jolla, CA, USA.
    Novel eicosanoid activators of PPAR gamma formed by RAW 264.7 macrophage cultures2002In: Advances in Experimental Medicine and Biology, ISSN 0065-2598, E-ISSN 2214-8019, Vol. 507, p. 343-349Article in journal (Refereed)
  • 143. Haussühl, Kirsten
    et al.
    Andersson, Bertil
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Cell biology.
    Adamska, Iwona
    A chloroplast DegP2 protease performs the primary cleavage of the photodamaged D1 protein in plant photosystem II2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4, p. 713-722Article in journal (Refereed)
    Abstract [en]

    Although light is the ultimate substrate in photosynthesis, it can also be harmful and lead to oxidative damage of the photosynthetic apparatus. The main target for light stress is the central oxygen-evolving photosystem II (PSII) and its D1 reaction centre protein. Degradation of the damaged D1 protein and its rapid replacement by a de novo synthesized copy represent the important repair mechanism of PSII crucial for plant survival under light stress conditions. Here we report the isolation of a single-copy nuclear gene from Arabidopsis thaliana, encoding a protease that performs GTP-dependent primary cleavage of the photodamaged D1 protein and hence catalysing the key step in the repair cycle in plants. This protease, designated DegP2, is a homologue of the prokaryotic Deg/Htr family of serine endopeptidases and is associated with the stromal side of the non-appressed region of the thylakoid membranes. Increased expression of DegP2 under high salt, desiccation and light stress conditions was measured at the protein level.

  • 144.
    Hedman, Christina
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Frystyk, Jan
    Medical Research Laboratories, Clinical Institute and Medical Department, Aarhus University Hospital, Aarhus, Denmark.
    Fridell, Karin
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Jönsson, Anna
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Flyvbjerg, Allan
    Medical Research Laboratories, Clinical Institute and Medical Department, Aarhus University Hospital, Aarhus, Denmark.
    Lindström, Torbjörn
    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.
    The IGF-system is not affected by a twofold change in protein intake in patients with type 1 diabetes2005In: Growth Hormone & IGF Research, ISSN 1096-6374, E-ISSN 1532-2238, Vol. 15, no 4, p. 304-310Article in journal (Refereed)
    Abstract [en]

    Objective In type 1 diabetes the circulating IGF-system is altered with low IGF-I and changes in levels of IGF-binding proteins (IGFBPs) which may be of importance for the development of diabetes complications. Our aim was to study if IGF-I, as supported by experimental data in animals, can be affected by dietary protein intake.

    Design and methods Twelve patients with type 1 diabetes, age 37.5 ± 10.0 years (mean ± SD), diabetes duration 20.1 ± 9.3 years and HbA1c 6.3 ± 0.6% were allocated to isocaloric diets with either low normal protein content (LNP), (10 E%; 0.9 g protein/kg body weight) or high normal protein content (HNP) (20 E%; 1.8 g protein/kg body weight) in an open randomised cross-over study. Each diet was taken for 10 days with a wash-out period of 11 days in between. Circulating levels of total and free IGF-I and -II, IGFBP-1, -2 and -3 and GH-binding protein (GHBP) as well as ghrelin were measured with validated in-house immunoassays.

    Results At day 10, urinary urea excretion was 320 ± 75 mmol/24 h during LNP diet compared with 654 ± 159 mmol/24 h during HNP diet (p < 0.001). There were no changes in body weight or glycaemic control between the diets. Fasting levels of total IGF-I were 121 ± 33 μg/L after LNP and 117 ± 28 μg/L after HNP diet (ns) and the corresponding concentrations of IGFBP-1 were 142(141) and 132(157) μg/L [median (IQR)] (ns). There were no differences in plasma concentrations of total IGF-II, free IGF-I and -II, IGFBP-3, GHBP and ghrelin, whereas a small difference was found for IGFBP-2 (302 ± 97 vs. 263 ± 66 μg/L; LNP vs. HNP; p < 0.04).

    Conclusions A twofold change of the dietary protein intake does not influence the altered circulating IGF-system in type 1 diabetes. In order to affect the IGF-system other interventions must be used.

  • 145.
    Hedman, Christina
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Frystyk, Jan
    Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark.
    Lindström, Torbjörn
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Chen, Jian-Wen
    Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark.
    Flyvbjerg, Allan
    Medical Research Laboratories, Aarhus University Hospital, Aarhus, Denmark.
    Ørskov, Hans
    Medical Research Laboratories, 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.
    Residual β-cell function more than glycemic control determines abnormalities of the insulin-like growth factor system in type 1 diabetes2004In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 89, no 12, p. 6305-6309Article in journal (Refereed)
    Abstract [en]

    The GH-IGF-I axis is disturbed in patients with type 1 diabetes. Our aim was to investigate whether abnormalities are found in patients in very good glycemic control and, if so, to estimate the role of residual β-cell function. Patients with hemoglobin A 1c (HbA 1c) less than 6% (reference range, 3.6-5.4%) were selected for the study. Twenty-two men and 24 women, aged 41.3 ± 13.8 yr (mean ± SD), with a diabetes duration of 17.8 ± 14.6 yr participated. Healthy controls (15 women and nine men), aged 41.3 ± 13.0 yr, were also studied. Overnight fasting serum samples were analyzed for HbA 1c, C peptide, free and total IGFs, IGF-binding proteins (IGFBPs), GH-binding protein, and IGFBP-3 proteolysis. HbA 1c was 5.6 ± 0.5% in patients and 4.4 ± 0.3% in controls. Total IGF-I was 148 ± 7 μg/liter in patients and 178 ± 9 μg/liter in controls (P < 0.001). Free IGF-I, total IGF-II, IGFBP-3, and GH-binding protein were lower, whereas IGFBP-1, IGFBP-1-bound IGF-I, and IGFBP-2 were elevated compared with control values. Patients with detectable C peptide (≥100 pmol/liter) had higher levels of total IGF-I, free IGF-I, and total IGF-II and lower levels of IGFBP-1 and IGFBP-2 than those with an undetectable C peptide level despite having identical average HbA 1c. IGFBP-3 proteolysis did not differ between patients and controls. Despite very good glycemic control, patients with type 1 diabetes and no endogenous insulin production have low free and total IGF-I. Residual β-cell function, therefore, seems more important for the disturbances in the IGF system than good metabolic control per se, suggesting that portal insulin delivery is needed to normalize the IGF system.

  • 146.
    Hedman, Christina
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Lindström, Torbjörn
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Arnqvist, Hans
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Direct comparison of insulin lispro and aspart shows small differences in plasma insulin profiles after subcutaneous injection in type 1 diabetes2001In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 24, p. 1120-1121Article in journal (Refereed)
    Abstract [en]

    No abstract available.

  • 147.
    Hedman, Christina
    et al.
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Orre-Pettersson, A-C
    Linköping University, Department of Medicine and Care, Internal Medicine. Linköping University, Faculty of Health Sciences.
    Lindström, Torbjörn
    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.
    Treatment with insulin lispro changes the insulin profile but does not affect the plasma concentrations of IGF-I and IGFBP-1 in type 1 diabetes2001In: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 55, no 1, p. 107-112Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE IGF-I levels in patients with type 1 diabetes without endogenous insulin production are low. Our aim was to examine whether the plasma insulin profile obtained by treatment with the insulin analogue lispro has a different effect on plasma concentrations of IGF-I and IGFBP-1 than that seen during treatment with conventional human insulin (regular insulin).

    DESIGN AND PATIENTS Twelve patients with type 1 diabetes, age 47·8 ± 2·4 years (mean ± SEM), body mass index 26·5 ± 1·0 kg/m2, diabetes duration 30·5 ± 3·2 years participated in this open label randomized cross-over study. IGF-I and IGFBP-1 levels were measured at the end of 6 weeks treatment with each insulin being administered by a continuous subcutaneous insulin infusion. IGF-I was measured fasting while IGFBP-1, free insulin and blood glucose were measured fasting and repeatedly after a morning meal preceded by an insulin bolus dose.

    RESULTS Lispro gave a marked insulin peak of 135 ± 20 pmol/l 50 minutes after injection. After an initial rapid rise, human regular insulin reached a plateau of approximately 50 pmol/l. The plasma free insulin area under the curve (AUC) from 0710 h to 0910 h was more than twice as large on lispro as on regular insulin (P = 0·01). Plasma IGF-I concentration was 78·8 ± 10·9 µg/l on lispro and 82·3 ± 10·5 µg/l on human regular insulin (not significant). AUC for IGFBP-1 did not show a significant difference even when divided from 0710 h to 0910 h and from 0930 h to 1430 h. Blood glucose AUC after administration of the bolus was significantly lower during treatment with lispro (P = 0·006) but glycosylated haemoglobin (HbA1c) was 6·4 ± 0·2% on both therapies.

    CONCLUSIONS Our results indicate that the effect of lispro on IGF-I and IGFBP-1 in patients with type 1 diabetes does not differ from that of human regular insulin.

  • 148.
    Hellberg, Carina
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    β2 integrin-induced signal transduction in granulocytic cells1997Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Neutrophil granulocytcs are highly mobile, phagocytic white blood cells whose motile functions are critically dependent on adhesion receptors belonging to the B2 integrin subfamily. These integrins are vital for neutrophil functions due to their ability to generate intracellular signals. The B2 integrin~rnediated phagocytosis of complement-opsonised particles is believed to be regulated by PKC. Particle engulfment has previously been found to be accompanied by accumulation of DG, a lipid known to activate PKC. In the present experiments, this DO originated mainly from PLD-mediated hydrolysis of phosphatidylcholinc. The subsequent activation of PKC was demonstrated by the ability of this kinase to phosphorylate the PKC~ specific substrate MARCKS. Moreover, PKC regulated the activity ofPLD in a positive way and thereby maintained its own activity. PKC has also been shown to phosphorylate the B2 subunit of the integrin. Although this phosphorylation does not regulate the avidity of the integrins, it may be needed for the induction of intracellular signals. However, antibody-induced ligation of B2 integrins did not alter the phosphorylation status of these integrins, indicating that this phosphorylation is not involved in the generation of second messengers. In contrast, pretreatment of cells with phorbol esters inhibited the tyrosine kinase activation and the subsequent rise in cytosolic free Ca2+ in a way that correlated with its ability to induce a phosphorylation of the B2 subunit of the integrin. Therefore, a PKC-mediated phosphorylation of this subunit may represent a mechanism for other receptors to regulate the signalling capacity of B2 integrins.

    The B2 integrin-induced ca2+ signal was shown to be due to both a release of ca2+ from intracellular stores and an influx across the plasma membrane, Moreover, integrin ligation induced a tyrosine phosphoryialion of PLCy2 accompanied by a small formation of Ins(1,4,5)P3, indicating that PLCy2 is responsible for the intracellular release of ca2+. Further evidence was provided by the findings that an activation tyrosine kinase(s) precedes, and is a prerequisite for, the rise in cytosolic free ca2+ concentration. The Src family tyrosine kinase Fgr is activated by B2 integrin-dependent neutrophil adhesion, and this occurs through mechanisms that are yet unidentified. The present results show that ligation of B2 integrins elicited a rapid and transient association of Fgr with B2 integrins. The tyrosine kinase inhibitor genistein did not affect this association, but it did prevent the subsequent dissociation. The interaction between these two proteins could represent an initial step in the B2 integrin-induced activation of Fgr, whereas the dissociation of Fgr from B2 integrins appears to be dependent on the activation of a tyrosinekinase, probably Fgr itself.

  • 149. Henricsson, Marianne
    et al.
    Nyström, Lennarth
    Blohmé, Göran
    Östman, Jan
    Kullberg, Carin
    Svensson, Maria
    Schölin, Anna
    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, Elisabeth
    Bolinder, Jan
    Eriksson, Jan
    Sundkvist, Göran
    The incidence of retinopathy 10 years after diagnosis in young adult people with diabetes: Results from the nationwide population-based Diabetes Incidence Study in Sweden (DISS)2003In: Diabetes Care, ISSN 0149-5992, E-ISSN 1935-5548, Vol. 26, no 2, p. 349-354Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE - To estimate the prevalence and severity of diabetic retinopathy (DR) 10 years after diagnosis in a nationwide population-based cohort study of young adult diabetic patients in Sweden. RESEARCH DESIGN AND METHODS - The Diabetes Incidence Study in Sweden (DISS) aims to register all incident cases of diabetes aged 15-34 years in Sweden. In 1987-1988, 806 cases were reported, and 627 (78%) of them were followed up with regard to retinopathy 8-10 years later. The assessment was based on retinal photographs in most cases (86%). RESULTS - Ten years after diagnosis, retinopathy was found in 247 patients (39%). The retinopathy was mild in 206 (33%), whereas 30 (4.8%) patients had moderate nonproliferative DR (NPDR) and 11 (1.8%) had proliferative DR (PDR). Patients with retinopathy had worse glycemic control during the years than patients without (HbA1c 8.1 ▒ 1.5% and 6.8 ▒ 1.2%, respectively, P < 0.001). In a Cox regression analysis, time to retinopathy was related to high HbA1c (P < 0.001) and high BMI (P = 0.001). Patients with type 2 diabetes had an increased prevalence of severe retinopathy (NPDR or PDR) compared with those with type 1 diabetes (14 of 93 [15%] versus no or mild 24 of 471 [5%], respectively, P < 0.001). CONCLUSIONS - Despite modern diabetes management, 39% of young adult diabetic patients developed retinopathy within the first 10 years of the disease. Nevertheless, compared with the prevalence of retinopathy (63%), after a similar duration of diabetes before the Diabetes Control and Complications Trial, this prevalence was clearly lower. Current treatment aimed to achieve strict glycemic control has reduced the risk for developing retinopathy.

  • 150.
    Herbertsson, Helena
    Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
    Studies on a novel cytosolic/nuclear binding protein for the eicosanoid 12(S)-hydroxyeicosatetraenoic acid1998Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Hydroxyeicosatetraenoic acids (HETEs) are lipoxygenase metabolites of arachidonic acid. HETEs were long considered to have few or no important biological function - a view that proved to be incorrect. These compounds have now been identified in many different types of cells, and they have been attributed several important actions, the molecular mechanisms of which are largely unclear. This thesis describes the discovery of a putative receptor for 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE).

    Specific high-affinity binding sites for 12(S)-HETE were detected in the cytosol (52%) and the nuclei (18%) of cells of the Lewis lung carcinoma (LLC) line. Similar l2(S)-HETE binding sites were detected in the cytosol of several other kinds of cells and in human platelets. Gel permeation chromatography and density gradient centrifugation indicated an apparent molecular weight of about 650 kDa and a sedimentation coefficient of20.5 S for the binding sites in the cytosol ofLLC cells. Treatment with ATP caused the 650 kDa component to dissociate into subunits. The actuall2(S)-HETE binding subunit was subsequently shown to have a molecular weight of about 50 kDa.

    The subcellular distribution of l2(S)-HETE binding sites in LLC cells was found to resemble the distribution of some nuclear receptors of the steroid hormone receptor superfamily. The untransformed glucocorticoid receptor has been reported to be located in cytosol in association with heat shock proteins 70 and 90, and Western blot analyses and immunoprecipitation revealed the same two proteins in the cytosolic 650 kDa l2(S)-HETE binding complex.

    A possible relationship to the steroid hormone receptor superfamily was also suggested by the observation that the 50 kDa l2(S)-HETE binding protein interacted with steroid receptor coactivator-l (SRC-l), which is known to be recruited to the site of transcriptional activity by several nuclear receptors. The interaction with SRC-1 occurred only when l2(S)-HETE was bound to its 50 kDa binding protein.

    In summary, the research presented in this thesis led to the discovery of a novel type of eicosanoid receptor. This binding site resembles the nuclear receptors for steroids and related compounds by virtue of its subcellular distribution, the inclusion of heat shock proteins in its binding complex, and its strictly ligand-dependent interaction with SRC-l.

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