| 1. |
- Benkestock, Kurt, et al.
(författare)
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Automated Nano-Electrospray Mass Spectrometry for Protein-Ligand Screening by Noncovalent Interaction Applied to Human H-FABP and A-FABP
- 2003
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Ingår i: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 8:3, s. 247-256
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Tidskriftsartikel (refereegranskat)abstract
- A method for ligand screening by automated nano-electrospray ionization mass spectrometry (nano-ESI/MS) is described. The core of the system consisted of a chip-based platform for automated sample delivery from a 96-well plate and subsequent analysis based on noncovalent interactions. Human fatty acid binding protein, H-FABP (heart) and A-FABP (adipose), with small potential ligands was analyzed. The technique has been compared with a previously reported method based on nuclear magnetic resonance (NMR), and excellent correlation with the found hits was obtained. In the current MS screening method, the cycle time per sample was 1.1 min, which is approximately 50 times faster than NMR for single compounds and approximately 5 times faster for compound mixtures. High reproducibility was achieved, and the protein consumption was in the range of 88 to 100 picomoles per sample. Furthermore, a novel protocol for preparation of A-FABP without the natural ligand is presented. The described screening approach is suitable for ligand screening very early in the drug discovery process before conventional high-throughput screens (HTS) are developed and/or used as a secondary screening for ligands identified by HTS.
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| 2. |
- Bernado, P, et al.
(författare)
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Combined use of NMR relaxation measurements and hydrodynamic calculations to study protein association. Evidence for tetramers of low molecular weight protein tyrosine phosphatase in solution
- 2003
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 125:4, s. 916-923
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Tidskriftsartikel (refereegranskat)abstract
- We describe a novel method for determining weak association constants of oligomeric protein complexes formed transiently under equilibrium conditions. This type of equilibrium process is recognized as being biologically important, but generally hard to study. Heteronuclear spin relaxation rates measured at multiple protein concentrations are analyzed using relaxation rates predicted from hydrodynamic calculations, yielding equilibrium constants and structural characterization of the protein complexes. The method was used to study the oligomerization equilibrium of bovine low molecular weight protein tyrosine phosphatase. X-ray structures of monomeric and dimeric forms of the protein have been reported previously. Using longitudinal and transverse 15N relaxation rates measured at four different protein concentrations, we detected the monomer, dimer, and a previously unknown tetramer and measured the dissociation constants of the equilibria involving these species. A comparison of experimental and predicted relaxation rates for individual backbone amide 15N spins enabled delineation of the tetramerization interface. The results suggest a novel concept for substrate modulation of enzymatic activity based on a "supramolecular proenzyme". The fast and reversible switching of the "supramolecular proenzyme" would have obvious advantages for the regulation of enzymes involved in cell signaling pathways.
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| 3. |
- Bjersand, Kathrine, 1972-, et al.
(författare)
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The clinical and prognostic correlation of HRNPM and SLC1A5 in pathogenesis and prognosis in epithelial ovarian cancer
- 2017
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:6
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: To evaluate the prognostic effect of the Heterogeneous nuclear ribonucleoprotein type M (HNRPM) and Solute carrier 1A5 (SLC1A5) in FIGO-stages I-II epithelial ovarian cancer.METHODS: A retrospective cohort study was designed to investigate the prognostic effect of HNRPM and SLC1A5, and the association with clinical-pathologic characteristics in 131 patients with FIGO-stages I-II epithelial ovarian cancer. Tissue microarrays were constructed and protein levels were assessed by immunohistochemistry (IHC).RESULTS: Positive HRNPM status was associated with positive staining for PUMA (P = 0.04), concomitant PUMA and p21 staining (P = 0.005), and VEGF-R2 (P = 0.003). Positive SLC1A5 staining was associated with positive staining of p27 (P = 0.030), PUMA (P = 0.039), concomitant PUMA and p27 staining, and VEGF-R2 (P = 0.039). In non-serous tumors (n = 72), the SLC1A5 positivity was associated with recurrent disease (P = 0.01). In a multivariable logistic regression analysis FIGO-stage (OR = 12.4), tumor grade (OR = 5.1) and SLC1A5 positivity (OR = 0.1) were independent predictive factors for recurrent disease. Disease-free survival (DFS) in women with SLC1A5-positive non-serous tumors was 92% compared with of 66% in patients with SLC1A5-negative non-serous tumors (Log-rank = 15.343; P = 0.008). In Cox analysis with DFS as endpoint, FIGO-stage (HR = 4.5) and SLC1A5 status (HR = 0.3) were prognostic factors.CONCLUSIONS: As the proteins HRNPM and SLC1A5 are associated with the cell cycle regulators p21 or p27, the apoptosis regulators PTEN and PUMA, and the VEGF-R2 it is concluded that both proteins have role in the pathogenesis of ovarian cancer. In patients with non-serous ovarian cancer SLC1A5 protects from recurrent disease, presumably by means of biological mechanisms that are unrelated to cytotoxic drug sensitivity.
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| 4. |
- Giordanetto, Fabrizio, et al.
(författare)
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Design of Selective sPLA2-X Inhibitor (-)-2-{2-[Carbamoyl-6-(trifluoromethoxy)-1 H-indol-1-yl]pyridine-2-yl}propanoic Acid
- 2018
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Ingår i: ACS Medicinal Chemistry Letters. - : American Chemical Society. - 1948-5875. ; 9:7, s. 600-605
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Tidskriftsartikel (refereegranskat)abstract
- A lead generation campaign identified indole-based sPLA2-X inhibitors with a promising selectivity profile against other sPLA2 isoforms. Further optimization of sPLA2 selectivity and metabolic stability resulted in the design of (-)-17, a novel, potent, and selective sPLA2-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (-)-17 was tested in an ApoE-/- murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA2-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (-)-17 did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis.
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| 5. |
- Skírnisdottir, Ingiridur, 1951-, et al.
(författare)
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Cell Cycle Regulator p27 Mediates Body Mass IndexEffects in Ovarian Cancer in FIGO-stages I-II
- 2019
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Ingår i: Cancer Genomics & Proteomics. - : International Institute of Anticancer Research. - 1109-6535 .- 1790-6245. ; 16:6, s. 443-450
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Tidskriftsartikel (refereegranskat)abstract
- Background/Aim: The aim of the present study was to evaluate the association between body mass index (BMI), the biomarker p27, and the clinical factors in FIGO-stages I-II ovarian cancer. Patients and Methods: A total of 128 patients with ovarian cancer were included in the study. For testing differences in univariate analyzes we used the Pearson's Chi-square test and the log-rank test. For multivariate analyses the logistic regression and Cox regression models were used with recurrent disease and disease free survival as endpoints, respectively. Results: Patients with BMI <= 25 kg/m(2) had a significantly better 5-year disease free survival compared with patients with BMI >25 kg/m(2) in the total series of patients (p=0.008), and in the series of patients (n=77) with non-serous tumors (p=0.047). Patients with p27-positive non-serous tumors had higher survival compared to patients with p27-negative non-serous tumors (p=0.020). Conclusion: The cell cycle regulator p27 mediates BMI effects in ovarian cancer in FIGO-stages I-II.
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| 6. |
- Skírnisdottir, Ingiridur, 1951-, et al.
(författare)
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Clinical significance of growth factor receptor EGFR and angiogenesis regulator VEGF‑R2 in patients with ovarian cancer at FIGO stages I-II.
- 2018
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 53:4, s. 1633-1642
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Tidskriftsartikel (refereegranskat)abstract
- The aim of the present retrospective cohort study was to investigate the prognostic effect of epidermal growth factor receptor (EGFR) and the angiogenesis regulator vascular endothelial growth factor receptor 2 (VEGF‑R2) on disease-free survival (DFS) rate and recurrent disease, and their association with clinicopathological characteristics in 131 patients with International Federation of Gynecology and Obstetrics (FIGO) stages I-II epithelial ovarian cancer. The techniques of tissue microar-rays and immunohistochemistry were used for the positive detection of the markers. The frequency of positive staining in tumors for EGFR was 24% and for VEGF‑R2 was 77%. Across the cohort, there was a total of 34/131 recurrences (26%) and the 5‑year DFS rate was 68%. In a multivariate logistic regression analysis with recurrent disease as the endpoint, FIGO stage (OR=9.7), type (I/II) of tumor (OR=3.0) and VEGF‑R2 status (OR=0.2) were all found to be independent predictive factors in the cohort of patients (n=131). For patients with non‑serous tumors (n=78), the FIGO stage (OR=76), type (I/II) of tumor (OR=44), EGFR status (OR=0.05) and VEGF‑R2 status (OR=0.008) were all significant and independent predictive factors. On comparing the four subgroups, in terms of concomitant EGFR and VEGF‑R2 status, in a survival analysis, the subgroup of patients (n=21) with concomitant positive expression of EGFR and VEGF‑R2 had a 5‑year DFS rate of 100%. Therefore, the prognostic effect of EGFR and VEGF‑R2 for recurrent disease and survival rates was confirmed by the above findings. Certain results in the present study were not in line with results from previous studies on the prognostic effect of EGFR and VEGF‑R2. An increasing number of preclinical and clinical observations have shown that the process of angiogenesis remains to be fully elucidated. Therefore, one of the challenges for future ovarian cancer investigations is to identify which biomarkers may be used as predictive and prognostic markers.
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| 7. |
- Skírnisdóttir, Ingiridur, 1951-, et al.
(författare)
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Differences in Clinical and Biological Features Between Type I and Type II Tumors in FIGO Stages I-II Epithelial Ovarian Carcinoma.
- 2015
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Ingår i: International Journal of Gynecological Cancer. - 1048-891X .- 1525-1438. ; 25:7, s. 1239-47
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The objective of this study was to compare immunohistochemical profile for the apoptosis regulators p53, C-MYC, bax, PUMA, and PTEN and the cell cycle regulatory proteins p21 and p27, as well as clinical factors between types I and II tumors.METHODS: In total, 131 patients in FIGO (International Federation of Gynecology and Obstetrics) stages I-II were divided into 2 groups of patients after type I tumors (n = 79) and type II tumors (n = 52). Differences in the immunohistochemical profile for the cell cycle-related proteins, detected by tissue microarrays and immune-histochemistry, were compared. For statistical tests, the Pearson χ test and the logistic regression model were used. All tests were 2-sided, and the level of statistical significance was P ≤ 0.05.RESULTS: In multivariate logistic regression analysis with recurrent disease as endpoint, FIGO stage (odds ratio [OR], 4.7), type I/II tumors (OR, 3.8), body mass index (BMI) (OR, 3.5), and p53 status (OR, 4.2) all were found to be independent predictive factors. In 2 different multivariate logistic regression analyses with type I/II tumors as endpoint, both p53p21 (OR, 2.9) and p27 status (OR, 3.0) were associated with type II tumors. Differently, C-MYC status (OR, 0.4) was associated with type I tumors. Furthermore, age (OR, 1.04), BMI (OR, 0.4), and recurrent disease (OR, 4.3) all were associated to type II tumors. In survival analysis, there was a trend (P = 0.054) toward better disease-free survival for patients with type I tumors.CONCLUSIONS: Concomitant positivity for p53 and negativity for p21, positivity for p27, and negativity for C-MYC in an epithelial ovarian tumor might strengthen the diagnostic option of type II tumor ovarian carcinoma. Patients with type II tumors were older, had lower BMI, and had more often recurrent disease than patients with type I tumors.
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| 8. |
- Skirnisdottir, Ingiridur, 1951-, et al.
(författare)
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Napsin A as a marker of clear cell ovarian carcinoma
- 2013
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 13:1, s. 524-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Clear cell carcinomas are aggressive tumors with a distinct biologic behaviour. In a genome-widescreening for genes involved in chemo-resistance, NAPA was over-expressed in cisplatin-resistant cells. The NAPA(protein) Napsin A was described to promote resistance to cisplatin by degradation of the tumor suppressor p53.Methods: Totally 131 patients were included in this study all in FIGO-stages I-II; 16 were clear cell tumors which were compared with 40 Type I tumors and 75 type II tumors according to the markers Napsin A, p21, p53 and p27 and some clinical features. For detection of the markers tissue microarrays and immunohistochemistry were used.Results: Positivity for Napsin A was detected in 12 (80%) out of the 15 clear cell tumors available for analysis compared with 3 (4%) out of the Type I and II tumors in one group (p < 0.001). Differences in p21 status, p53 status, and p21 + p53- status were striking when clear cell tumors were compared with Type I, Type II, and Type I and II tumors in one group, respectively. The p21 + p53-status was associated to positive staining of Napsin A (p = 0.0015)and clear cell morphology (p = 0.0003). In two separate multivariate logistic regression analyses with Napsin A as endpoint both clear cell carcinoma with OR = 153 (95% C.I. 21–1107); (p < 001) and p21 + p53- status with OR = 5.36(95% C.I. 1.6-17.5); (p = 0.005) were independent predictive factors. ROC curves showed that AUC for Napsin A alone was 0.882, for p21 + p53- it was 0.720 and for p21 + p53-Napsin A + AUC was 0.795. Patients with clear cell tumors had lower (p = 0.013) BMI than Type I patients and were younger (p = 0.046) at diagnosis than Type II patients. Clearcell tumors had a higher frequency (p = 0.039) of capsule rupture at surgery than Type I and II tumors.Conclusions: Positivity of Napsin A in an epithelial ovarian tumor might strengthen the morphological diagnosis ofclear cell ovarian carcinoma in the process of differential diagnosis between clear cell ovarian tumors and other histological subtypes.
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| 9. |
- Skírnisdottir, Ingiridur, 1951-, et al.
(författare)
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The relationship of the angiogenesis regulators VEGF-A, VEGF-R1 and VEGF-R2 to p53 status and prognostic factors in epithelial ovarian carcinoma in FIGO-stages I-II
- 2016
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Ingår i: International Journal of Oncology. - : Spandidos Publications. - 1019-6439 .- 1791-2423. ; 48:3, s. 998-1006
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Tidskriftsartikel (refereegranskat)abstract
- The aim of this study was to evaluate prognostic effect of the angiogenesis regulators VEGF-R1, VEGF-R2 and VEGF-A for recurrent disease and disease-free survival (DFS), and their relation to the apoptosis regulator p53, in 131 patients with FIGO-stages I-II with epithelial ovarian cancer. For the detection of positivity of the markers the techniques of tissue microarrays and immunohistochemistry (IHC) were used. In tumors the frequency of positive staining for VEGF-R1 was 19%, for VEGF-R2 and VEGF-A, it was 77 and 70%, respectively. Positivity for p53 was detected in 25% of tumors. The total number of recurrences in the complete series was 34 out of 131 (26%) and 5-year disease-free survival (DFS) was 68%. Positive staining for VEGF-A (P=0.030), VEGF-R2 (P=0.011) and p53 (P=0.015) was found more frequently in type II tumors than in type I tumors. Patients with VEGF-R1 negative tumors had worse (P=0.021) DFS compared to patients with VEGF-R1 positive tumors. In two multivariate Cox analyzes with DFS as endpoint, FIGO-stage (HR=3.8), VEGF-R2 status (HR=0.4) and p53 status (HR=2.3), all were significant and independent prognostic factors. When the variables VEGF-R2 and p53 were replaced with the new variable VEGF-R2(+)p53(-/)other three combinations in one group, it was found that patients from that subgroup had 86% reduced risk of dying in disease (HR=0.24). Findings above, confirmed relationship between VEGF-R2 and VEGF-A and p53, respectively, with regard to recurrent disease and survival. Some findings from the present study are different from results from previous studies on the regulation of angiogenesis. Despite many trials with anti-angiogenic agents in the front line of ovarian cancer have shown to be positive for progression-free survival, no one has demonstrated an impact on overall survival. Therefore, one of the greatest challenges in ovarian cancer research, is to discover predictive and prognostic biomarkers.
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| 10. |
- Åkerud, Tomas, et al.
(författare)
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Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: A model for changes in dynamics upon target binding
- 2002
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Ingår i: Journal of Molecular Biology. - 1089-8638. ; 322:1, s. 137-152
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Tidskriftsartikel (refereegranskat)abstract
- Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K-d = 1.5(+/-0.1) mM and an off-rate on the order of 10(4) s(-1).H-1 and N-15 NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. N-15 relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer. (C) 2002 Elsevier Science Ltd. All rights reserved.
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| 11. |
- Åkerud, Tomas
(författare)
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Protein Dynamics Studied by NMR. Kinetics of the Adipocyte Fatty Acid-Binding Protein and Oligomerisation of the Low Molecular Weight Protein Tyrosine Phosphatase.
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nuclear magnetic resonance spectroscopy (NMR) has been used to determine the kinetics of ligand binding to the adipocyte fatty acid-binding protein (A-FABP), as well as the oligomerisation and inter- and intramolecular dynamics of the low molecular weight-protein tyrosine phosphatase (LMW-PTP) in solution. NMR spectroscopy is sensitive to dynamic processes on timescales ranging from picoseconds to days. Using 15N isotope-enriched protein samples, dynamic processes have been probed with high spatial resolution, by monitoring each amide group in the protein. The kinetics of ligand binding to A-FABP was studied by 15N-longitudinal relaxation-exchange experiments. Using competitive binding experiments, it proved feasible to determine the kinetic rate constants of ligands with higher affinity than otherwise possible using protein NMR. The study included the important natural ligand oleate, a fatty acid mimetic denoted BVT.1961 (3-[4-hydroxy-3-isopropylphenyl]-propionic acid) and a fatty acid mixture consisting of myristate, palmitate, palmitoleate, cis-9,10-methylene-hexadecanoate, cis- vaccenoate and stearate. The measured off-rates ranged between 0.8 and 5 s–1 and the dissociation constants between 0.5 and 10 m M, with the lowest values for oleate and the highest for BVT.1961. The oligomerisation of LMW-PTP was extensively characterised using concentration dependent chemical shifts, as well as concentration dependent longitudinal and transverse relaxation rates. The chemical shift changes were confined to a region of the protein surface that corresponds to the interface between the two molecules in the dimeric crystal structure of of LMW-PTP. The oligomerisation state and dissociation constants for the oligomers were determined by fitting hydrodynamic models to the concentration dependent relaxation rates. The model that fitted the experimental data best was a monomer-dimer-tetramer equilibrium of LMW-PTP.
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