How does displacement of albumin-bound tryptophan cause sustained increases in the free tryptophan concentration in plasma and 5-hydroxytryptamine synthesis in brain?
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ABSTRACT:
SUBMITTER: Salter M
PROVIDER: S-EPMC1133271 | biostudies-other | 1989 Aug
REPOSITORIES: biostudies-other
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Project description:Signal transduction based on fluorescence is one of the most common optical aptasensors for small molecules. Sensors with a number of unique features including high sensitivity, low cost, and simple operation can be constructed easily. However, the label-free fluorescent approach is limited to synthetic dyes that bind strongly to the aptamer sequence and result in a diminished sensor operation with high detection limits. In this study, we report the use of curcumin as a fluorescent probe to signal aptamer/small target binding events. A substantial enhancement in curcumin's fluorescent emission was observed when bound into the grooves of vitamin D3 (VTD3) binding aptamer, as an example. However, the introduction of the target molecule causes the aptamer to undergo a conformational change that favors complexing the target molecule over binding the curcumin dye. The sensor was able to detect VTD3 down to 1 fM concentration in buffer solutions and extracted blood samples, operate at a wide dynamic range, and discriminate against potential biological interfering molecules including VTD2. The operation of the curcumin based fluorescent sensor is at least six orders of magnitude more sensitive than a VTD3 sensor constructed with the synthetic dye SYBR Green I. The generality of the reported label-free approach was applied with a previously isolated 75-mer bisphenol-A (BPA) aptamer, confirming that the reported sensing strategy is not confined on a particular aptamer sequence. Our work not only reports a novel sensor format for the detection of small molecules, but also serves fluorescent sensor's most pressing need being novel fluorophores for multiplex targets detection.
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Project description:BackgroundL-Tryptophan (L-Trp) derivatives such as 5-hydroxytryptophan (5-HTP) and 5-hydroxytryptamine (5-HT), N-Acetyl-5-hydroxytryptamine and melatonin are important molecules with pharmaceutical interest. Among, 5-HT is an inhibitory neurotransmitter with proven benefits for treating the symptoms of depression. At present, 5-HT depends on plant extraction and chemical synthesis, which limits its mass production and causes environmental problems. Therefore, it is necessary to develop an efficient, green and sustainable biosynthesis method to produce 5-HT.ResultsHere we propose a one-pot production of 5-HT from L-Trp via two enzyme cascades for the first time. First, a chassis cell that can convert L-Trp into 5-HTP was constructed by heterologous expression of tryptophan hydroxylase from Schistosoma mansoni (SmTPH) and an artificial endogenous tetrahydrobiopterin (BH4) module. Then, dopa decarboxylase from Harminia axyridis (HaDDC), which can specifically catalyse 5-HTP to 5-HT, was used for 5-HT production. The cell factory, E. coli BL21(DE3)△tnaA/BH4/HaDDC-SmTPH, which contains SmTPH and HaDDC, was constructed for 5-HT synthesis. The highest concentration of 5-HT reached 414.5 ± 1.6 mg/L (with conversion rate of 25.9 mol%) at the optimal conditions (substrate concentration,2 g/L; induced temperature, 25℃; IPTG concentration, 0.5 mM; catalysis temperature, 30℃; catalysis time, 72 h).ConclusionsThis protocol provided an efficient one-pot method for converting. L-Trp into 5-HT production, which opens up possibilities for the practical biosynthesis of natural 5-HT at an industrial scale.
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Project description:The lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL, compared with -non-preferring CBA, mice is caused by a decrease in circulating tryptophan availability to the brain secondarily to a higher liver tryptophan pyrrolase activity associated with a higher circulating corticosterone concentration. Activity or expression of liver tryptophan pyrrolase and/or their induction by glucocorticoids may be important biological determinants of predisposition to alcohol consumption.
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Project description:Nanoporous alumina membranes have become a ubiquitous biosensing platform for a variety of applications and aptamers are being increasingly utilized as recognition elements in protein sensing devices. Combining the advantages of the two, we report label-free sensitive detection of human ?-thrombin by an aptamer-functionalized nanoporous alumina membrane using a four-electrode electrochemical cell. The sensor response to ?-thrombin was determined in the presence of a high concentration (500??M) of human serum albumin (HSA) as an interfering protein in the background. The sensor sensitivity was also characterized against ?-thrombin, which is a modified ?-thrombin lacking the aptamer binding epitope. The detection limit, within an appreciable signal/noise ratio, was 10?pM of ?-thrombin in presence of 500??M HSA. The proposed scheme involves the use of minimum reagents/sample preparation steps, has appreciable response in presence of high concentrations of interfering molecules and is readily amenable to miniaturization by association with existing-chip based electrical systems for application in point-of-care diagnostic devices.
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Project description:Background and aimsSerotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models.Approach and resultsWhile in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2-/- ) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2-/- - mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2-/- - mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2-/- mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2-/- mice, respectively. 5HT levels increase in Mdr2-/- mice and in PSC human patients compared to their controls and decrease in serum of Mdr2-/- mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2-/- mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls.ConclusionsModulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.
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Project description:Tween (polysorbate) 20 and 80 are surfactants used for the development of parenteral protein drugs, due to their beneficial safety profile and stabilisation properties. To elucidate the mechanism by which Tween 20 and 80 stabilise proteins in aqueous solutions, either by a "direct" protein to surfactant interaction and/or by an interaction with the protein film at the air-water interface, we used spectroscopic (Infrared Reflection Absorption Spectroscopy, IRRAS) and microscopic techniques (Brewster Angle Microscopy, BAM) in combination with surface pressure measurements. To this end, the impact of both types of Tweens with regard to the displacement of the protein from the air-water interface was studied. As a model protein, human serum albumin (HSA) was used. The results for the displacement of the adsorbed HSA films by Tweens 20 and 80 can partially be understood on the basis of an orogenic displacement mechanism, which depends on the critical surface pressure of the adsorbed protein film. With increasing concentration of Tween in the sub-phase, BAM images showed the formation of different domain morphologies. IRRA-spectra supported the finding that at high protein concentration in the sub-phase, the protein film could not be completely displaced by the surfactants. Comparing the impact of both surfactants, we found that Tween 20 adsorbed faster to the protein film than Tween 80. The adsorption kinetics of both Tweens and the speed of protein displacement increased with rising surfactant concentration. Tween 80 reached significant lower surface pressures than Tween 20, which led to an incomplete displacement of the observed HSA film.
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Project description:In this study, we investigated the relationship between tryptophan-5-hydroxytryptamine metabolism, depressive disorder, and gastrointestinal dysfunction in rats after myocardial infarction. Our goal was to elucidate the physiopathologic bases of somatic/psychiatric depression symptoms after myocardial infarction. A myocardial infarction model was established by permanent occlusion of the left anterior descending coronary artery. Depression-like behavior was evaluated using the sucrose preference test, open field test, and forced swim test. Gastric retention and intestinal transit were detected using the carbon powder labeling method. Immunohistochemical staining was used to detect indoleamine 2,3-dioxygenase expression in the hippocampus and ileum. High-performance liquid chromatography with fluorescence and ultraviolet detection determined the levels of 5-hydroxytryptamine, its precursor tryptophan, and its metabolite 5-hydroxyindoleacetic acid in the hippocampus, distal ileum, and peripheral blood. All data were analyzed using one-way analyses of variance. Three weeks after arterial occlusion, rats in the model group began to exhibit depression-like symptoms. For example, the rate of sucrose consumption was reduced, the total and central distance traveled in the open field test were reduced, and immobility time was increased, while swimming, struggling and latency to immobility were decreased in the forced swim test. Moreover, the gastric retention rate and gastrointestinal transit rate were increased in the model group. Expression of indoleamine 2,3-dioxygenase was increased in the hippocampus and ileum, whereas 5-hydroxytryptamine metabolism was decreased, resulting in lower 5-hydroxytryptamine and 5-hydroxyindoleacetic acid levels in the hippocampus and higher levels in the ileum. Depressive disorder and gastrointestinal dysfunction after myocardial infarction involve abnormal tryptophan-5-hydroxytryptamine metabolism, which may explain the somatic, cognitive, and psychiatric symptoms of depression commonly observed after myocardial infarction. Peripheral 5-hydroxytryptamine is an important substance in the gut-brain axis, and its abnormal metabolism is a critical finding after myocardial infarct.
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Project description:Introduction: The drug-plasma protein interaction is a fundamental issue in guessing and checking the serious drug side effects related with other drugs. The purpose of this research was to study the interaction of cephalexin with bovine serum albumin (BSA) and displacement reaction using site probes. Methods: The interaction mechanism concerning cephalexin (CPL) with BSA was investigated using various spectroscopic methods and molecular modeling method. The binding sites number, n, apparent binding constant, K, and thermodynamic parameters, ΔG0, ΔH0, and ΔS0 were considered at different temperatures. To evaluate the experimental results, molecular docking modeling was calculated. Results: The distance, r=1.156 nm between BSA and CPL were found in accordance with the Forster theory of non-radiation energy transfer (FRET) indicating energy transfer occurs between BSA and CPL. According to the binding parameters and ΔG0= negative values and ΔS0= 28.275 j mol-1K-1, a static quenching process is effective in the CPL-BSA interaction spontaneously. ΔG0 for the CPL-BSA complex obtained from the docking simulation is -28.99 kj mol-1, which is close to experimental ΔG of binding, -21.349 kj mol-1 that indicates a good agreement between the results of docking methods and experimental data. Conclusion: The outcomes of spectroscopic methods revealed that the conformation of BSA changed during drug-BSA interaction. The results of FRET propose that CPL quenches the fluorescence of BSA by static quenching and FRET. The displacement study showed that phenylbutazon and ketoprofen displaced CPL, indicating that its binding site on albumin is site I and Gentamicin cannot be displaced from the binding site of CPL. All results of molecular docking method agreed with the results of experimental data.
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2023-05-30 | GSE233714 | GEO