Summary
Aim : The gastrointestinal transit of sequentially administered capsules was investigated in relation to the availability of fluid along the intestinal lumen by magnetic resonance imaging.
...Methods : Water‐sensitive magnetic resonance imaging was performed on 12 healthy subjects during fasting and 1 h after a meal. Specifiable non‐disintegrating capsules were administered at 7, 4 and 1 h prior to imaging.
Results : While food intake reduced the mean fluid volumes in the small intestine (105 ± 72 mL vs. 54 ± 41 mL, P < 0.01) it had no significant effect on the mean fluid volumes in the colon (13 ± 12 mL vs. 18 ± 26 mL). The mean number of separated fluid pockets increased in both organs after meal (small intestine: 4 vs. 6, P < 0.05; large intestine: 4 vs. 6, P < 0.05). The distribution of capsules between the small and large intestine was strongly influenced by food (colon: 3 vs. 17 capsules, P < 0.01).
Conclusions : The results show that fluid is not homogeneously distributed along the gut, which likely contributes to the individual variability of drug absorption. Furthermore, transport of fluid and solids through the ileocaecal valve is obviously initiated by a meal‐induced gastro‐ileocaecal reflex.
The enzymatic synthesis of nucleoside analogues has been shown to be a sustainable and efficient alternative to chemical synthesis routes. In this study, dihalogenated nucleoside analogues were ...produced by thermostable nucleoside phosphorylases in transglycosylation reactions using uridine or thymidine as sugar donors. Prior to the enzymatic process, ideal maximum product yields were calculated after the determination of equilibrium constants through monitoring the equilibrium conversion in analytical-scale reactions. Equilibrium constants for dihalogenated nucleosides were comparable to known purine nucleosides, ranging between 0.071 and 0.081. To achieve 90% product yield in the enzymatic process, an approximately five-fold excess of sugar donor was needed. Nucleoside analogues were purified by semi-preparative HPLC, and yields of purified product were approximately 50% for all target compounds. To evaluate the impact of halogen atoms in positions 2 and 6 on the antiproliferative activity in leukemic cell lines, the cytotoxic potential of dihalogenated nucleoside analogues was studied in the leukemic cell line HL-60. Interestingly, the inhibition of HL-60 cells with dihalogenated nucleoside analogues was substantially lower than with monohalogenated cladribine, which is known to show high antiproliferative activity. Taken together, we demonstrate that thermodynamic calculations and small-scale experiments can be used to produce nucleoside analogues with high yields and purity on larger scales. The procedure can be used for the generation of new libraries of nucleoside analogues for screening experiments or to replace the chemical synthesis routes of marketed nucleoside drugs by enzymatic processes.
Ruminococcin A (RumA) is a lanthipeptide with high activity against pathogenic clostridia and is naturally produced by the strict anaerobic bacterium
E1, isolated from human intestine. Cultivating
E1 ...is challenging, limiting high-quality production, further biotechnological development and therapeutic exploitation of RumA. To supply an alternative production system, the gene encoding RumA-modifying enzyme (RumM) and the gene encoding the unmodified precursor peptide (preRumA) were amplified from the chromosome of
E1 and coexpressed in
. Our results show that the ruminococcin-A lanthionine synthetase RumM catalyzed dehydration of threonine and serine residues and subsequently installed thioether bridges into the core structure of a mutant version of preRumA (preRumA
). These modifications were achieved when the peptide was expressed as a fusion protein together with green fluorescence protein (GFP), demonstrating that a larger attachment to the N-terminus of the leader peptide does not obstruct
processivity of RumM in modifying the core peptide. The leader peptide serves as a docking sequence which the modifying enzyme recognizes and interacts with, enabling its catalytic role. We further investigated RumM catalysis in conjunction with the formation of complexes observed between RumM and the chimeric GFP fusion protein. Results obtained suggested some insights into the catalytic mechanisms of class II lanthipeptide synthetases. Our data further indicated the presence of three thioether bridges, contradicting a previous report whose findings ruled out the possibility of forming a third ring in RumA. Modified preRumA
was activated
by removing the leader peptide using trypsin and biological activity was achieved against
ATCC 6633. A production yield of 6 mg of pure modified preRumA
per liter of
culture was attained and considering the size ratio of the leader-to-core segments of preRumA
, this amount would generate a final yield of approximately 1-2 mg of active RumA when the leader peptide is removed. The yield of our system exceeds that attainable in the natural producer by several 1000-fold. The system developed herein supplies useful tools for product optimization and for performing
peptide engineering to generate new analogs with superior anti-infective properties.
The measurement of values of apparent equilibrium constants
' for enzyme-catalyzed reactions involve a substantial number of critical details, neglect of which could lead to systematic errors. Here, ...interferences, impurities in the substances used, and failure to achieve equilibrium are matters of substantial consequence. Careful reporting of results is of great importance if the results are to have archival value. Thus, attention must be paid to the identification of the substances, specification of the reaction(s), the conditions of reaction, the definition of the equilibrium constant(s) and standard states, the use of standard nomenclature, symbols, and units, and uncertainties. This document contains a general discussion of various aspects of these equilibrium measurements as well as STRENDA (
andards for
eporting
zymology
ta) recommendations regarding the measurements and the reporting of results.
Well-characterized small molecules enable the study of cell processes and facilitate target validation. Here, we describe a high-content multiplex screen to investigate cell viability over 48 h, ...which can be combined with investigating phenotypic features, such as tubulin binding and mitochondrial content, as initial cellular quality control of diverse compounds. The protocol is on a live-cell basis and easily adaptable and scalable. It details cell preparation, compound handling, plate layout configuration, image acquisition with the CQ1, and data analysis using the CellPathfinder software.
For complete details on the use and execution of this protocol, please refer to Tjaden et al. (2022).
Display omitted
•A fast and flexible multiplex assay for compound annotation•Evaluate cell properties in live-cell mode•Machine learning techniques to optimize high-content data evaluation•Easily adaptable and scalable for different phenotypic features
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Well-characterized small molecules enable the study of cell processes and facilitate target validation. Here, we describe a high-content multiplex screen to investigate cell viability over 48 h, which can be combined with investigating phenotypic features, such as tubulin binding and mitochondrial content, as initial cellular quality control of diverse compounds. The protocol is on a live-cell basis and easily adaptable and scalable. It details cell preparation, compound handling, plate layout configuration, image acquisition with the CQ1, and data analysis using the CellPathfinder software.
Efficient reaction monitoring is crucial for data acquisition in kinetic and mechanistic studies. However, for conversions of nucleosides to their corresponding nucleobases, as observed in ...enzymatically catalyzed nucleoside phosphorylation reactions, the current analytical arsenal does not meet modern requirements regarding cost, speed of analysis and high throughput. Herein, we present a UV/Vis spectroscopy-based assay employing an algorithm for spectral unmixing in a 96-well plate format. The algorithm relies on fitting of reference spectra of nucleosides and their bases to experimental spectra and allows determination of nucleoside/nucleobase ratios in solution with high precision. The experimental procedure includes appropriate dilution of a sample into aqueous alkaline solution, transfer to a multi-well plate, measurement of a UV/Vis spectrum and subsequent in silico spectral unmixing. This enables data collection in a high-throughput fashion and reduces costs compared to state-of-the-art HPLC analyses by approximately 5-fold while being 20-fold faster and offering comparable precision. Additionally, the method is robust regarding dilution and sample transfer errors as it only considers spectral form and not absolute intensity. It can be applied to all natural nucleosides and nucleobases and even unnatural ones as demonstrated by several examples.
The COVID-19 pandemic has highlighted the need for FAIR (Findable, Accessible, Interoperable, and Reusable) data more than any other scientific challenge to date. We developed a flexible, ...multi-level, domain-agnostic FAIRification framework, providing practical guidance to improve the FAIRness for both existing and future clinical and molecular datasets. We validated the framework in collaboration with several major public-private partnership projects, demonstrating and delivering improvements across all aspects of FAIR and across a variety of datasets and their contexts. We therefore managed to establish the reproducibility and far-reaching applicability of our approach to FAIRification tasks.
Nucleoside analogs represent a class of important drugs for cancer and antiviral treatments. Nucleoside phosphorylases (NPases) catalyze the phosphorolysis of nucleosides and are widely employed for ...the synthesis of pentose‐1‐phosphates and nucleoside analogs, which are difficult to access via conventional synthetic methods. However, for the vast majority of nucleosides, it has been observed that either no or incomplete conversion of the starting materials is achieved in NPase‐catalyzed reactions. For some substrates, it has been shown that these reactions are reversible equilibrium reactions that adhere to the law of mass action. In this contribution, we broadly demonstrate that nucleoside phosphorolysis is a thermodynamically controlled endothermic reaction that proceeds to a reaction equilibrium dictated by the substrate‐specific equilibrium constant of phosphorolysis, irrespective of the type or amount of NPase used, as shown by several examples. Furthermore, we explored the temperature‐dependency of nucleoside phosphorolysis equilibrium states and provide the apparent transformed reaction enthalpy and apparent transformed reaction entropy for 24 nucleosides, confirming that these conversions are thermodynamically controlled endothermic reactions. This data allows calculation of the Gibbs free energy and, consequently, the equilibrium constant of phosphorolysis at any given reaction temperature. Overall, our investigations revealed that pyrimidine nucleosides are generally more susceptible to phosphorolysis than purine nucleosides. The data disclosed in this work allow the accurate prediction of phosphorolysis or transglycosylation yields for a range of pyrimidine and purine nucleosides and thus serve to empower further research in the field of nucleoside biocatalysis.
Background and methods
The antiepileptic drug carbamazepine is known to be an inducer of cytochrome P450 (CYP) 3A4 after binding to the nuclear pregnane X receptor. To evaluate whether it also ...regulates the multidrug transporter proteins P‐glycoprotein (P‐gp) and multidrug resistance protein MRP2 in humans, duodenal expression of multidrug resistance gene MDR1 messenger ribonucleic acid (mRNA) and MRP2 mRNA, content of P‐gp and MRP2, and disposition of the nonmetabolized P‐gp substrate talinolol after intravenous (30 mg) and long‐term oral administration (100 mg for 19 days) were assessed in 7 healthy subjects (age, 23–35 years; body weight, 64–93 kg) before and after comedication of carbamazepine (600 mg for 14–18 days).
Results
Carbamazepine medication was associated with increased urinary excretion of D‐glucaric acid and induction of carbamazepine elimination. Creatinine clearance was not affected. Duodenal expression of both MDR1 mRNA and MRP2 mRNA and the MPR2 protein was significantly induced, whereas the P‐gp content was not affected. MDR1 mRNA expression and MPR2 mRNA expression were correlated (r = 0.873, P < .001). After carbamazepine, metabolic clearance of intravenous talinolol was significantly increased. Residual clearance was significantly decreased in dependence on MDR1 mRNA expression (r = −0.647, P = .012) and MRP2 mRNA expression (r = −0.613, P = .020). Oral absorption of talinolol was significantly lower after carbamazepine comedication (53.2% ± 15.5% versus 62.1% ± 13.0%, P = .018), and renal clearance and metabolic clearance were significantly increased, correlated in each case with MDR1 mRNA (r = 0.612, P = .020, and r = 0.554, P = .040, respectively) and MRP2 mRNA (r = 0.596, P = .025, and r = 0.565, P = .035, respectively).
Conclusions
Aside from induction of CYP3A4, carbamazepine acts as an inducer of intestinal MDR1 mRNA, MRP2 mRNA, and MRP2 protein content.
Clinical Pharmacology & Therapeutics (2004) 76, 192–200; doi: 10.1016/j.clpt.2004.04.011
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