Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-¹³C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.     

Does small-sided-games’ court area influence metabolic,perceptual, and physical performance parameters of young elite basketball players?

The purpose of this study was to investigate the effect of court size on physiological responses and physical performance of young elite basketball players. Twelve male basketball players (18.6 ± 0.5 years; 88.8 ± 14.5 kg; 192.6 ± 6.5 cm) from an under-19 team performed two small-sided games (matches) with different court areas (28x15 m and 28x9 m; 28x15 and 28x9 protocols). The number of players (3x3) was kept the same in each protocol. The players performed a repeated-sprint ability (RSA) test before and after each match. Blood lactate concentration was collected before (pre) and after (post) the matches, and the session rating of perceived exertion (session-RPE) was determined 30 minutes after the match. Best and mean time in the RSA test were not different between the 28x15 and the 28x9 match protocols (p > 0.05). A significant difference was observed for lactate concentration from pre- to post-match (p < 0.05) in both protocols (28x15 and 28x9); however, there was no significant interaction between protocols. A similar session-RPE mean score (28x15: 7.2 ± 1.4 and 28x9: 6.6 ± 1.4) was detected for both protocols (p > 0.05, ES=0.41). In summary, the results of the current study suggest that the different court areas induced similar responses. Although there was no significant difference in effort perception, players tended to perceive a greater effort in the larger court size.     

Structure of the human lung cytochrome P450 2A13

The human lung cytochrome P450 2A13 (CYP2A13) activates the nicotine-derived procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into DNA-altering compounds that cause lung cancer. Another cytochrome P450, CYP2A6, is also present in human lung, but at much lower levels. Although these two enzymes are 93.5% identical, CYP2A13 metabolizes NNK with much lower K(m) values than does CYP2A6. To investigate the structural differences between these two enzymes the structure of CYP2A13 was determined to 2.35A by x-ray crystallography and compared with structures of CYP2A6. As expected, the overall CYP2A13 and CYP2A6 structures are very similar with an average root mean square deviation of 0.5A for the Calpha atoms. Like CYP2A6, the CYP2A13 active site cavity is small and highly hydrophobic with a cluster of Phe residues composing the active site roof. Active site residue Asn(297) is positioned to hydrogen bond with an adventitious ligand, identified as indole. Amino acid differences between CYP2A6 and CYP2A13 at positions 117, 300, 301, and 208 relate to different orientations of the ligand plane in the two protein structures and may underlie the significant variations observed in binding and catalysis of many CYP2A ligands. In addition, docking studies suggest that residues 365 and 366 may also contribute to differences in NNK metabolism.     

Detection and characterization of individual intermolecular bonds using optical tweezers

The development of scanning probe techniques has made it possible to examine protein-protein interactions at the level of individual molecular pairs. A calibrated optical tweezers, along with immunoglobulin G (IgG)-coated polystyrene microspheres, has been used to detect individual surface-linked Staphylococcus protein A (SpA) molecules and to characterize the strength of the noncovalent IgG-SpA bond. Microspheres containing, on average, less than one IgG per contact area were held in the optical trap while an SpA-coated substrate was scanned beneath them at a distance of approximately 50 nm. This geometry allows the trapped bead to make contact with the surface, from bond formation to rupture, and results in an enhancement of the force applied to a bond due to leverage supplied by the bead itself. Experiments yielded median single-bond rupture forces from 25 to 44 pN for IgG from four mammalian species, in general agreement with predictions based on free energies of association obtained from solution equilibrium constants.     

Surgically induced cryptorchidism-related degenerative changes in spermatogonia are associated with loss of cyclic adenosine monophosphate-dependent phosphodiesterases type 4 in abdominal testes of rats

The present study was undertaken to investigate the role of phosphodiesterase type 4 (PDE4) enzymes in cryptorchidism-induced apoptosis of the germ cells. Regulation of expression of PDE4 enzymes was studied in the abdominal and scrotal testes of surgically induced cryptorchid rats for 10, 20, and 30 days. In some cases orchidopexy was performed after 30 days of cryptorchidism, and rats were allowed to recover for an additional 50 days. Upon histological examination, marked degenerative changes in the epithelial lining of the seminiferous tubules within abdominal testes were observed compared with contralateral control or age-matched sham-operated rats. These changes included degeneration of some spermatogonia, apoptosis of the secondary spermatocytes, incomplete spermatogenesis, and lack of spermatozoa in the lumen. In contrast, contralateral scrotal testes exhibited normal histology. Significant improvement in the regeneration of spermatogonia was observed in rats after 50 days of recovery following orchidopexy. Immunocytochemical examination suggested the presence of PDE4A in germ cells while PDE4B was predominantly expressed on somatic cells. Western blotting using PDE4 subtype-selective antibodies showed the presence of two PDE4A variants (a 109-kDa PDE4A8 and a previously uncharacterized 88-kDa PDE4A variant) and two PDE4B (78-kDa PDE4B2 and 66-kDa PDE4B variant) bands. In unilaterally cryptorchid animals, the abdominal testis showed a time-dependent decrease in both PDE4A8 and 88-kDa PDE4A variants. In contrast, the expression of 66-kDa PDE4B was markedly increased in a time-dependent fashion in abdominal testes of cryptorchid rats. Animals surgically corrected for cryptorchidism and allowed to recover for 50 days exhibited normal expression of both PDE4A and PDE4B variants compared with aged-matched, sham-operated controls. In conclusion, this study suggests that down-regulation of PDE4A variants in cryptorchid testes may play an important role in the degeneration of spermatogonia and increased apoptotic activity in the germ cells.     

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.     

Effect of two and five days of creatine loading on anaerobic working capacity in women

The purpose of this study was to determine the effects of 2 and 5 days of Cr loading on anaerobic working capacity (AWC) using the critical power (CP) test in women. Ten physically active women randomly received 2 treatments separated by a 5 week washout period: (A) 18 g dextrose as placebo (PL) or (B) 5.0 g Cr + 18 g dextrose taken 4 times per day for 5 days. Following a familiarization trial, each subject completed the CP test at baseline and following 2 and 5 days of supplementation. The PL resulted in no significant changes in AWC following supplementation; however, Cr increased AWC by 22.1% after 5 days of loading (p < 0.05). There was a significant main effect for body weight (BW), however, there was no significant increase in BW due to Cr supplementation. These results suggest that Cr supplementation is effective for increasing AWC in women following 5 days of loading without an associated increase in BW.     

Structure of mammalian cytochrome P450 2C5 complexed with diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding

The structure of the anti-inflammatory drug diclofenac bound in the active site of rabbit microsomal cytochrome P450 2C5/3LVdH was determined by X-ray crystallography to 2.1 A resolution. P450 2C5/3LVdH and the related enzyme 2C5dH catalyze the 4'-hydroxylation of diclofenac with apparent K(m) values of 80 and 57 microM and k(cat) values of 13 and 16 min(-1), respectively. Spectrally determined binding constants are similar to the K(m) values. The structure indicates that the pi-electron system of the dichlorophenyl moiety faces the heme Fe with the 3'- and 4'-carbons located 4.4 and 4.7 A, respectively, from the Fe. The carboxyl moiety of the substrate is hydrogen bonded to a cluster of waters that are also hydrogen bonded to the side chains of N204, K241, S289, and D290 as well as the backbone of the protein. The proximity of the diclofenac carboxylate to the side chain of D290 together with an increased binding affinity at lower pH suggests that diclofenac is protonated when bound to the enzyme. The structure exhibits conformational changes indicative of an adaptive fit to the substrate reflecting both the hydration and size of the substrate. These results indicate how structurally diverse substrates are recognized by drug-metabolizing P450 enzymes.     

Effects of in vitro production on horse embryo morphology,cytoskeletal characteristics,and blastocyst capsule formation

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.