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9 záznamů v Medvik Filtry

Článek

Measurement of protein synthesis: in vitro comparison of (68)Ga-DOTA-puromycin, [ (3)H]tyrosine, and 2-fluoro-[ (3)H]tyrosine

Recent Results in Cancer Research. 2013 ; 194 () : 269-83.

Recent Results Cancer Res
ISSN 0080-0015
Medvik
Zdroj

AIM: Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. METHODS: DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur. RESULTS: No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. SUMMARY: Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.

Článek

The interaction of standard oxime reactivators with hemicholinium-3 sensitive choline carriers

Toxicology letters. 2012 ; 212 (3) : 315-319.

Toxicol Lett
ISSN 1879-3169
Medvik
Zdroj

Organophosphorus poisoning manifests as a cholinergic syndrome due to an inhibition of acetylcholinesterase. It is treated symptomatically by anticholinergics and oxime reactivators are used as causal antidotes. Reactivators possess a complex mechanism of action and interact at various levels of the cholinergic transmission. The aim of this study was to investigate the effect of standard oxime reactivators (HI-6, obidoxime, trimedoxime, methoxime and pralidoxime) on the hemicholinium-3 sensitive carriers, which are involved in the high-affinity choline uptake (HACU) transport, a key regulatory step in the synthesis of acetylcholine. The activity of the carriers was estimated in vitro on hippocampal synaptosomes using the substrate (3H)-choline and the competitive inhibitor (3H)-hemicholinium-3. Furthermore, the effect of the reactivators on the fluidity of hippocampal membranes was assessed. All tested compounds, except methoxime, showed an acute inhibitory effect on the carriers, however, only at μM concentrations. Trimedoxime showed the highest potency to inhibit HACU among all tested compounds (I(max) 62%, IC(50)=3 μM). All compounds, except HI-6, influenced also a membrane fluidity in the region of the hydrophilic heads of phospholipid bilayer, nevertheless, only methoxime was able to penetrate more deeply into the hydrocarbon core. We suggest that the direct interaction of oxime reactivators with the carrier protein (HI-6 and trimedoxime) and/or the changes in carrier conformation mediated by alterations in membrane fluidity (trimedoxime, obidoxime and pralidoxime) could occur here. The influence of reactivators on the carriers could be unfavorable in the case of their prolonged administration in vivo. From this point of view, the application of methoxime appears to be the best.

Článek

Gastric submucosal microdialysis in the detection of rat stomach ischemia--a comparison of the 3H2O efflux technique with metabolic monitoring

Physiological measurement. 2010 ; 31 (10) : 1355-68. [pub] 20100823

Physiol Meas
ISSN 1361-6579
Medvik
Zdroj

Microdialysis has been utilized for nutritive blood flow measurements, but both the advantages and disadvantages of various approaches have not been evaluated in parallel in the stomach yet. Our aim was to compare the (3)H(2)O efflux technique with biochemical monitoring during temporary celiac artery occlusion in anesthetized rats. Microdialysis probes were implanted in the gastric submucosa and perfused with (3)H(2)O; samples were analyzed for β-activity, glucose, lactate, pyruvate and glycerol. Gastric mucosa and plasma were subjected to morphometry and analysis of myeloperoxidase, total thiols and lactatdehydrogenase. The most dramatic responses to ischemia were observed in lactate/pyruvate and lactate/glucose (%) ratios (6.1-9.3×, p < 0.0001); the changes in (3)H(2)O efflux and glycerol were less pronounced (1.1-1.7×, p < 0.0001 and < 0.01, respectively). (3)H(2)O efflux correlated best with the lactate/glucose ratio and glucose alone (r = 0.693 and -0.681, respectively, p < 0.0001). A correlation was also found between plasma lactatdehydrogenase and relative glycerol release (r = 0.600, p < 0.05). Myeloperoxidase, lactatdehydrogenase and histology score were increased by ischemia/reperfusion (0.06-0.12 nkat g(-1), p < 0.05, 0.26-0.44 nkat g(-1), p < 0.05 and 1.79-2.33, p < 0.05, respectively), macroscopy and plasma thiols remained unchanged. Microdialysis is useful in monitoring gastric ischemia, metabolic monitoring being superior to the (3)H(2)O efflux technique. The results question the efficacy of the utilized model to produce standardized major gastric damage.