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- Ahlsén, Göran, et al.
(författare)
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Resistance profiles of cyclic and linear inhibitors of HIV-1 protease
- 2002
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Ingår i: Antiviral Chemistry & Chemotherapy. - 0956-3202 .- 2040-2066. ; 13:1, s. 27-37
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Tidskriftsartikel (refereegranskat)abstract
- Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.
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| 2. |
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| 3. |
- Ahlsén, Maria
(författare)
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Gene expression in human skeletal muscle : effects of activity, fibre type and inheritance for diabetes
- 2006
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- One fundamental underlying risk factor for the development of non-insulin dependent (type 2) diabetes mellitus is insulin resistance, which 80-90% of all type 2 diabetic patients suffer from. Risk factors for insulin resistance are for example obesity, surgery, severe illness, spinal cord injury, and pregnancy. Too high energy intake and physical inactivity are direct causes of the increasing prevalence of overweight and obesity. Exercise prevents or delays the onset of type 2 diabetes. A series of beneficial effects of exercise are seen e.g. increased muscle mass, increased oxygen uptake, increased insulin sensitivity and glucose transport. Skeletal muscle represents 55% of the total body weight and is the major organ for insulinstimulated glucose uptake. The increased uptake of glucose during exercise is mediated via alternative, insulin-independent mechanisms e.g. through activated protein kinase (AMPK). A cohort of healthy glucose tolerant first-degree relatives of type 2 diabetic patients was studied to determine whether early metabolic and molecular defects contribute to insulin resistance in skeletal muscle. Skeletal muscle strips from relatives and control subjects were incubated in vitro in the absence or presence of increasing concentrations of insulin. Glucose transport, Akt phosphorylation, AS 160 phosphorylation, and GLUT4 expression were assessed. Insulin-stimulated glucose transport at a maximal insulin concentration was reduced in the relatives. Insulin increased Akt and AS 160 phosphorylation in a dose-dependent manner, with similar responses between the groups. Gene expression of some of the key transcriptional factors and co-regulators of mitochondrial biogenesis PGC-1alpha, PGC-1beta, PPARdelta, NRF-1 and UCP-3 was assessed. All these genes showed similar expression when corrected to either Pactin or GAPDH, indicating that disturbances in mitochondrial function do not precede development of insulin resistance and type 2 diabetes. PPARalpha, PPARdelta, PGC-1alpha, PGC-1beta, calcineurin Aalpha and calcineurin Abeta have also been implicated in the regulation of skeletal muscle fibre type distribution, as well as mitochondrial biogenesis. mRNA levels of these genes was determined in skeletal muscle biopsies from elite athletes with a significantly (P<0.05) higher portion of type I fibres compared to sedentary controls. mRNA expression of these genes was also assessed in spinal cord injured subjects who have lost almost all of their type 1 fibres. PPARalpha, PPARdelta, PGC-1alpha, PGC-1beta mRNA correlated to fibre types in skeletal muscle, whereas calcineurin mRNA expression was unrelated. Finally, gene expression pattern of AMPK alpha, beta and gamma isoforms in skeletal muscle from ablebodied subjects and spinal cord injured individuals was established. The effect of an eight week training study was determined on gene expression in spinal cord injured subjects after cycling seven days a week. Expression of AMPK also differed depending on time since injury. In summary, this thesis work has shown that exercise promotes a more oxidative phenotype in skeletal muscle, with increased glucose uptake. This is also seen in skeletal muscle that has been unused for several years. Exercised muscle, with a more oxidative phenotype has increased expression of several genes important for mitochondrial biogenesis and function. No alteration in mRNA expression of these genes is noted in skeletal muscle from subjects with a genetic predisposition for metabolic disease prior to onset of clinical symptoms.
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| 4. |
- Ahlsén, Maria
(författare)
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Insulin-like growth factor binding protein-3 : structure and function
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Insulin-like growth factor (IGF) -I and -II share structural homology with insulin. Their receptors and intracellular signaling are similar. Consequently, the biological actions of IGFs and insulin are overlapping. Insulin is secreted on demand depending on e.g. blood glucose while IGFs are continuously produced. IGFs are bound to specific IGF binding proteins (IGFBPs) with affinities higher than to the signaling receptor. The focus of this thesis is IGFBP-3, the major carrier of IGFs in the circulation. We have studied posttranslational modification of IGFBP-3, such as proteolysis and glycosylation, and their impact on IGF binding and actions. The IGFBPs have IGF binding sites in the N- and C-terminal domains. Prior to this thesis the binding domains in IGFBP-3 had not been fully characterized. We studied amino acids in the N-terminal hydrophobic binding pocket of IGFBP-3 and their importance for IGF binding. Directed mutations of residues I56, L80 and L81 with substitution for glycine resulted in single mutations with markedly decreased IGF binding. Double L80 and L81 mutations yielded an IGFBP-3 mutant with almost complete loss of affinity for IGF-I and -II and the triple mutant did not have detectable IGF binding. The ability of the IGFBP-3 mutants to inhibit IGF-Istimulated phosphorylation of its receptor reflected the IGF binding affinity. In states of insulin resistance, including pregnancy and diabetes, IGFBP-3 is proteolyzed. However, the identity of the proteolytic fragment was unknown prior to this work. We isolated a 30 kDa IGFBP-3 fragment from human pregnancy serum and identified, by N- and C-terminal amino acid sequence analysis and mass spectrometry, a fragment corresponding to residues 1-212 of the intact protein. The same fragment was also isolated from non-pregnancy serum in which it coexists with intact IGFBP-3. Using biosensor technology, we determined IGF binding kinetics. Compared to intact IGFBP-3, (1-212)IGFBP-3 had 11-fold lower affinity for IGF-I and 4-fold lower affinity for IGF-II entirely due to faster dissociations. Glycosylation of IGFBP-3 does not affect IGF binding. We demonstrated that glycosylation of IGFBP-3 protects against proteolysis in pregnancy and diabetes serum in vitro. Thus, glycosylation may indirectly affect IGF binding. In vitro cleavage of non-glycosylated IGFBP-3 occurs at four different major sites, none of which is identical to the cleavage site generating the naturally occurring (1-212)IGFBP-3 fragment in pregnancy. These data suggest that proteolysis of endogenous glycosylated IGFBP-3 takes place outside the circulation. Finally, we demonstrated that intact IGFBP-3 inhibited insulin stimulated glucose uptake in isolated skeletal muscles from mouse and rat. This effect, previously reported in vivo and in fat cells, was specific to fast twitch EDL and independent of Akt (protein kinase B) phosphorylation. This thesis has contributed to the current understanding of IGF interactions with IGFBP-3. Glycosylation of IGFBP-3 protects from serum proteolysis, suggesting that the dominant (1-212)IGFBP-3 species in pregnancy is produced by tissue proteases. The inhibitory effects of IGFBP-3 on insulin stimulated glucose uptake indicate that, in addition to reducing IGF binding, IGFBP-3 proteolysis may have other effects on insulin and IGF signaling. These changes may be essential to the pregnant woman in supporting optimal fetal growth.
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| 5. |
- Kostovski, Emil, et al.
(författare)
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Altered content of AMP-activated protein kinase isoforms in skeletal muscle from spinal cord injured subjects
- 2013
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Ingår i: American Journal of Physiology. Endocrinology and Metabolism. - Bethesda, MD : American Physiological Society. - 0193-1849 .- 1522-1555. ; 305:9, s. E1071-E1080
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Tidskriftsartikel (refereegranskat)abstract
- AMP-activated protein kinase (AMPK) is a pivotal regulator of energy homeostasis. Although downstream targets of AMPK are widely characterized, the physiological factors governing isoform expression of this protein kinase are largely unknown. Nerve/contractile activity has a major impact on the metabolic phenotype of skeletal muscle, therefore likely to influence AMPK isoform expression. Spinal cord injury represents an extreme form of physical inactivity, with concomitant changes in skeletal muscle metabolism. We assessed the influence of longstanding and recent spinal cord injury on protein abundance of AMPK isoforms in human skeletal muscle. We also determined muscle fiber type as a marker of glycolytic or oxidative metabolism. In subjects with longstanding complete injury, protein abundance of the AMPKγ3 subunit, as well as myosin heavy chain (MHC) IIa and IIx, were increased, whereas abundance of the AMPKγ1 subunit and MHC I were decreased. Similarly, abundance of AMPKγ3 and MHC IIa proteins were increased, whereas AMPKα2, -β1, and -γ1 subunits and MHC I abundance was decreased during the first year following injury, reflecting a more glycolytic phenotype of the skeletal muscle. However, in incomplete cervical lesions, partial recovery of muscle function attenuated the changes in the isoform profile of AMPK and MHC. Furthermore, exercise training (electrically stimulated leg cycling) partly normalized mRNA expression of AMPK isoforms. Thus, physical activity affects the relative expression of AMPK isoforms. In conclusion, skeletal muscle abundance of AMPK isoforms is related to physical activity and/or muscle fiber type. Thus, physical/neuromuscular activity is an important determinant of isoform abundance of AMPK and MCH. This further underscores the need for physical activity as part of a treatment regimen after spinal cord injury to maintain skeletal muscle metabolism. © 2013 the American Physiological Society.
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