Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.     

The uptake of [14C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

Imidazopyrimidines,potent inhibitors of p38 MAP kinase

The MAP kinase p38 is implicated in the release of the pro-inflammatory cytokines TNF-alpha and IL-1 beta. Inhibition of cytokine release may be a useful treatment for inflammatory conditions such as rheumatoid arthritis and Crohn's disease. A novel series of imidazopyrimidines have been discovered that potently inhibit p38 and suppress the production of TNF-alpha in vivo.     

Folding and assembly of large macromolecular complexes monitored by hydrogen-deuterium exchange and mass spectrometry

Recent advances in protein mass spectrometry (MS) have enabled determinations of hydrogen deuterium exchange (HDX) in large macromolecular complexes. HDX-MS became a valuable tool to follow protein folding, assembly and aggregation. The methodology has a wide range of applications in biotechnology ranging from quality control for over-expressed proteins and their complexes to screening of potential ligands and inhibitors. This review provides an introduction to protein folding and assembly followed by the principles of HDX and MS detection, and concludes with selected examples of applications that might be of interest to the biotechnology community.     

Inhibitors of unactivated p38 MAP kinase

Inhibition of the p38 map kinase pathway has been shown to be beneficial in the treatment of inflammatory diseases. The first class of potent p38 kinase inhibitors was the pyridinylimidazole compounds from SKB. Since then several pyridinylimidazole-based compounds have been shown to inhibit activated p38 kinase in vitro and in vivo. We have developed a novel series of pyridinylimidazole-based compounds, which potently inhibit the p38 pathway by binding to unactivated p38 kinase and only weakly inhibiting activated p38 kinase activity in vitro.     

Detection of the antimicrobial peptide gene in different Amaranthus species

Using primers to amplify the gene AMP2 in Amaranthus caudatus, we found the gene to be present in seven other species of the Amaranthus genus (A. albus, A. cruentus, A. blitum, A. hybridus, A. hypochondriacus, A. retroflexus and A. tricolor), in which it had not been described previously. The PCR products were sequenced and it was established that all the sequences were identical, except for two polymorphisms. These single nucleotide polymorphisms occurred at nucleotide positions 45 and 246. This exchange of one nucleotide for another was manifested in an amino acid change in both cases. Due to the fact that both polymorphisms lay outside the region encoding the chitin-binding peptide domain, which is crucial for antimicrobial peptide function, they will not likely affect the proper functioning of the peptide. With the exception of the above-mentioned polymorphisms, all sequences were identical to the sequence of the AMP2 gene that codes for the A. caudatus Ac-AMP2 (antimicrobial peptide isolated from Amaranthus caudatus seeds). The detection of sequences with high degree of sequence similarity to A. caudatus AMP2 gene leads us to the assumption that an antimicrobial peptide could also be produced by other amaranth species.     

The uptake of [C]glucose into rabbit ear cartilage proteogylcans isolated by differential extraction and by collagenase digestion

Rabbit ear cartilage was incubated with [¹⁴C]glucose and proteoglycans were extracted from the crushed cartilage by differential extraction. The extraction was performed sequentially with 0.15, 0.45 and 1.0 M NaCl, followed by 0.1 M acetic acid. The tissue residue was digested with collagenase and finally with papain. Each extractant, with the exception of 0.1 M acetic acid, released uronic acid containing material into the solution. The extractable proteoglycans represented together about 20% of the whole tissue uronate, the proteoglycans released by collagenase treatment accounted for a further 30%. The rest was insoluble and was released by papain. The highest specific radioactivity was found in the 0.15 M NaCl extract, decreasing progressively in subsequent extracts. The lowest specific activity was found in the chondroitin sulfate released from the tissue residue by papain. Radioactivity in the collagen-associated proteoglycans was comparable to the radioactivity of 1.0 M NaCl extracts. All salt extractable proteoglycans were retarded by Sepharose 6B and were found to be heterogeneous in size and rate of precursor uptake. Chondroitin sulfate released from each proteoglycan fraction was also heterogeneous in size and metabolic activity.     

The metabolic heterogeneity of rabbit ear cartilage chondroitin sulphate

The only glycosaminoglycans that can be isolated from the ear cartilage of 2-month-old rabbits are chondroitin 4-sulphate and chondroitin 6-sulphate. These chondroitin sulphates exhibit molecular-weight polydispersity when isolated from tissue by papain digestion. The chondroitin sulphate is metabolically heterogeneous in that radioactive precursors [(14)C]glucose or [(35)S]sulphate are preferentially incorporated into the higher-molecular-weight polymers both in vivo and in vitro. No transfer of radioactivity from the high-molecular-weight chondroitin sulphate to the low-molecular-weight chondroitin sulphate was seen during 15 days in vivo. It is suggested that there are at least two pools of proteoglycan in the tissue. One of these pools is metabolically active whereas the other is not.     

Notes on methods used in research of natural populations

The statistical method applied byKon?alová (1972) to examine the frequency of some taxonomic characters is discussed and another interpretation of the data presented is proposed.     

The effects of 12-week progressive strength training on strength,functional capacity,metabolic biomarkers,and serum hormone concentrations in healthy older women: morning versus evening training

ABSTRACT

Previous findings suggest that performing strength training (ST) in the evening may provide greater benefit for young individuals. However, this may not be optimal for the older population. The purpose of this study was to compare the effects of a 12-week ST program performed in the morning vs. evening on strength, functional capacity, metabolic biomarker and basal hormone concentrations in older women. Thirty-one healthy older women (66 ± 4 years, 162 ± 4 cm, 75 ± 13 kg) completed the study. Participants trained in the morning (M) (07:30, n = 10), in the evening (E) (18:00, n = 10), or acted as a non-training control group (C) (n = 11). Both intervention groups performed whole-body strength training with 3 sets of 10–12 repetitions with 2–3 minutes rest between sets. All groups were measured before and after the 12-week period with; dynamic leg press and seated-row 6-repetition maximum (6-RM) and functional capacity tests (30-second chair stands and arm curl test, Timed Up and Go), as well as whole-body skeletal muscle mass (SMM) (kg) and fat mass (FM-kg, FM%) assessed by bioelectrical impedance (BIA). Basal blood samples (in the intervention groups only) taken before and after the intervention assessed low-density lipoprotein (LDL-C), high-density lipoprotein (HDL-C), blood glucose (GLU), triglycerides (TG), high-sensitive C-reactive protein (hsCRP) concentrations and total antioxidant status (TAS) after a 12 h fast. Hormone analysis included prolactin (PRL), progesterone (P) estradiol (ESTR), testosterone (T), follicle stimulating hormone (FSH), and luteinizing hormone (LH). While C showed no changes in any variable, both M and E significantly improved leg press (+ 46 ± 22% and + 21 ± 12%, respectively; p < 0.001) and seated-row (+ 48 ± 21% and + 42 ± 18%, respectively; p < 0.001) 6-RM, as well as all functional capacity outcomes (p < 0.01) due to training. M were the only group to increase muscle mass (+ 3 ± 2%, p < 0.01). Both M and E group significantly (p < 0.05) decreased GLU (–4 ± 6% and –8 ± 10%, respectively), whereas significantly greater decrease was observed in the E compared to the M group (p < 0.05). Only E group significantly decreased TG (–17 ± 25%, p < 0.01), whereas M group increased (+ 15%, p < 0.01). The difference in TG between the groups favored E compared to M group (p < 0.01). These results suggest that short-term “hypertrophic” ST alone mainly improves strength and functional capacity performance, but it influences metabolic and hormonal profile of healthy older women to a lesser extent. In this group of previously untrained older women, time-of-day did not have a major effect on outcome variables, but some evidence suggests that training in the morning may be more beneficial for muscle hypertrophy (i.e. only M significantly increased muscle mass and had larger effect size (M: g = 2 vs. E: g = 0.5).