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991.
Dunja Urbančič Anita Kotar Alenka Šmid Marko Jukič Stanislav Gobec Lars-Göran Mårtensson Janez Plavec Irena Mlinarič-Raščan 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):182-190
Background
Methylation driven by thiopurine S-methylatransferase (TPMT) is crucial for deactivation of cytostatic and immunosuppressant thiopurines. Despite its remarkable integration into clinical practice, the endogenous function of TPMT is unknown.Methods
To address the role of TPMT in methylation of selenium compounds, we established the research on saturation transfer difference (STD) and 77Se NMR spectroscopy, fluorescence measurements, as well as computational molecular docking simulations.Results
Using STD NMR spectroscopy and fluorescence measurements of tryptophan residues in TPMT, we determined the binding of selenocysteine (Sec) to human recombinant TPMT. By comparing binding characteristics of Sec in the absence and in the presence of methyl donor, we confirmed S-adenosylmethionine (SAM)-induced conformational changes in TPMT. Molecular docking analysis positioned Sec into the active site of TPMT with orientation relevant for methylation reaction. Se-methylselenocysteine (MeSec), produced in the enzymatic reaction, was detected by 77Se NMR spectroscopy. A direct interaction between Sec and SAM in the active site of rTPMT and the formation of both products, MeSec and S-adenosylhomocysteine, was demonstrated using NMR spectroscopy.Conclusions
The present study provides evidence on in vitro methylation of Sec by rTPMT in a SAM-dependant manner.General significance
Our results suggest novel role of TPMT and demonstrate new insights into enzymatic modifications of the 21st amino acid. 相似文献992.
Anton A. Polyansky Eduard V. Bocharov Amélie I. Velghe Andrey S. Kuznetsov Olga V. Bocharova Anatoly S. Urban Alexander S. Arseniev Bojan Zagrovic Jean-Baptiste Demoulin Roman G. Efremov 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):82-95
Single-point mutations in the transmembrane (TM) region of receptor tyrosine kinases (RTKs) can lead to abnormal ligand-independent activation. We use a combination of computational modeling, NMR spectroscopy and cell experiments to analyze in detail the mechanism of how TM domains contribute to the activation of wild-type (WT) PDGFRA and its oncogenic V536E mutant. Using a computational framework, we scan all positions in PDGFRA TM helix for identification of potential functional mutations for the WT and the mutant and reveal the relationship between the receptor activity and TM dimerization via different interfaces. This strategy also allows us design a novel activating mutation in the WT (I537D) and a compensatory mutation in the V536E background eliminating its constitutive activity (S541G). We show both computationally and experimentally that single-point mutations in the TM region reshape the TM dimer ensemble and delineate the structural and dynamic determinants of spontaneous activation of PDGFRA via its TM domain. Our atomistic picture of the coupling between TM dimerization and PDGFRA activation corroborates the data obtained for other RTKs and provides a foundation for developing novel modulators of the pathological activity of PDGFRA. 相似文献
993.
Heracek J Hampl R Richard H Hill M Martin H Starka L Luboslav S Sachova J Jana S Kuncova J Jitka K Eis V Vaclav E Urban M Michael U Mandys V Vaclav M 《Steroids》2007,72(4):375-380
Androgens are considered to play a substantial role in pathogenesis of both benign prostatic hyperplasia (BPH) and prostate cancer. The importance of determination of androgen levels in tissue and serum for cancer progression and prognosis has been poorly understood. The aim of study was to find out hormonal differences in both diseases, their correlations between intraprostatic and serum levels and predicted value of their investigation. Testosterone, dihydrotestosterone, androstenedione and also epitestosterone were determined in prostate tissue from 57 patients who underwent transvesical prostatectomy for BPH and 121 patients after radical prostatectomy for prostate cancer. In 75 subjects with cancer and 51 with BPH the serum samples were analyzed for testosterone, dihydrotestosterone and SHBG. Significantly higher intraprostatic androgen concentrations, i.e. 8.85+/-6.77 versus 6.44+/-6.43 pmol/g, p<0.01 for dihydrotestosterone, and 4.61+/-7.02 versus 3.44+/-4.53 pmol/g, p<0.05 for testosterone, respectively, were found in patients with prostate cancer than in BPH. Higher levels in cancer tissue were found also for epitestosterone. However, no differences were found in serum levels. Highly significant correlations occurred between all pairs of intraprostatic androgens and also epitestosterone as well as between serum testosterone and dihydrotestosterone (p<0.001) in both BPH and cancer groups. Correlation was not found between corresponding tissue and serum testosterone and dihydrotestosterone, either in benign or cancer samples. The results point to importance of intraprostatic hormone levels for evaluation of androgen status of patients, contrasting to a low value of serum hormone measurement. 相似文献
994.
In a previous study, we showed that operators of radiofrequency (RF) plastic sealers, RF operators (n = 35) had a lower heart rate during nighttime compared to a control group (n = 37). We have analyzed the heart rate variability (HRV) on the same group of people to better understand the possible underlying rhythm disturbances. We found a significantly increased total HRV and very low frequency (VLF) power during nighttime among the RF operators compared to a control group. Together with our previous finding of a significantly lower heart rate during nighttime among the RF operators compared to the controls, this finding indicates a relative increase in parasympathetic cardiac modulation in RF operators. This could in turn be due to an adaptation of the thermoregulatory system and the cardiac autonomic modulation to a long-term low-level thermal exposure in the RF operators. 相似文献
995.
996.
Mirshamsi S Olsson M Arnelo U Kinsella JM Permert J Ashford ML 《Regulatory peptides》2007,141(1-3):19-24
A number of hormones, including leptin, have been shown to inhibit food intake in humans and animals. Analogues of 3-guanidinopropionic acid have also been found to reduce total food intake, but their mechanisms of action have not been well studied. The present study investigated the effects of intracerebroventricular infusion of the analogue BVT.3531 on food intake, meal pattern, and body weight in rats during 7 days. Single channel recordings from arcuate neurons and insulinoma cells were used to determine the effects of BVT.3531 on K(ATP) activity. Data analysis showed that BVT.3531 significantly decreased body weight and food intake, primarily by reducing meal size. BVT.3531 activated K(ATP) channels in cell-attached recordings from insulin-secreting cells and rat arcuate neurons but had no effect on K(ATP) channel activity in inside-out membrane patches from either cell type. BVT.3531 did not alter the firing rate or K(+) channel activity of arcuate neurons devoid of K(ATP). The study suggests that small molecules capable of mimicking the effects of leptin on food intake and body weight may utilize output mechanisms similar to those of leptin to elicit changes in arcuate neuron excitability. 相似文献
997.
A comparison of all known mammalian CYP1A sequences identifies nineteen sequence regions that are conserved within all 1A1s or within all 1A2s but at the same time systematically differ between any 1A1 and any 1A2. The purpose of this study was to explore links between these specific CYP1A sequence signatures and substrate specificity shift through the kinetic analysis of combinatorial variants of increasing complexity. The less complex variants correspond to multiple mutations within a short segment of their sequence. The more complex variants correspond to mosaic P450s recombining 1A1 and 1A2 sequences (up to 5 crossovers per sequence). Fifty-eight such functional CYP1A variants and parental wild-type enzymes were expressed in yeast and assayed with 7-alkoxyresorufins and ethoxyflurorescein ethyl ester as substrates. Observed kinetic data were analyzed by multivariate statistical analyses and hierarchical clustering in order to highlight correlations and identify potential sequence-activity relationships within the three-dimensional function space investigated. Several variants are outliers in these representations and show a redistribution of their substrate specificity compared to wild-type CYP1As. Some combinations of sequence elements were identified that significantly discriminate between 1A1 and 1A2 for these three substrates. The comparison of this combinatorial approach with previous results of site-directed mutagenesis is discussed. 相似文献
998.
Notch signaling induces SKP2 expression and promotes reduction of p27Kip1 in T-cell acute lymphoblastic leukemia cell lines 总被引:4,自引:0,他引:4
Dohda T Maljukova A Liu L Heyman M Grandér D Brodin D Sangfelt O Lendahl U 《Experimental cell research》2007,313(14):3141-3152
In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 intracellular domain (ICD) to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels. In contrast, pharmacologically blocking Notch signaling reversed this situation and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression. 相似文献
999.
Wachi H Sato F Nakazawa J Nonaka R Szabo Z Urban Z Yasunaga T Maeda I Okamoto K Starcher BC Li DY Mecham RP Seyama Y 《The Biochemical journal》2007,404(1):63-70
AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39-43 residue long Abeta (amyloid-beta)-peptide. The most abundant species, Abeta(1-40) and Abeta(1-42), are both present within senile plaques, but Abeta(1-42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Abeta-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Abeta(1-40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Abeta(1-42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Abeta(1-40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two beta-strands connected with a bend. This protection pattern partly resembles the pattern found in Abeta(1-42) fibrils, but the Abeta(1-40) fibrils display a significantly increased protection for the N-terminal residues Phe4-His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37-Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture. 相似文献
1000.
Wiesing U 《Bioethics》2007,21(7):398-405
DEFINITION OF THE PROBLEM: The regulation of residents' work hours involves several ethical conflicts which need to be systematically analysed and evaluated. ARGUMENTS AND CONCLUSION: The most important ethical principle when regulating work hours is to avoid the harm resulting from the over-work of physicians and from an excessive division of labour. Additionally, other ethical principles have to be taken into account, in particular the principles of nonmaleficence and beneficence for future patients and for physicians. The article presents arguments for balancing the relevant ethical principles and analyses the structural difficulties that occur unavoidably in any regulation of the complex activities of physicians. 相似文献