Creatine kinase isoenzyme activity during and after an ultra-distance (200 km) run

It is commonly assumed that creatine kinase (CK) activity in plasma is related to the state of an inflammatory response at 24-48 h, and also it has shown biphasic patterns after a marathon run. No information is available on CK isoenzymes after an ultra-marathon run. The purpose of the present study is to examine the CK isoenzymes after a 200 km ultra-marathon run and during the subsequent recovery. Blood samples were obtained during registration 1 2 h before the 200-km race and during the race at 100 km, 150 km and at the end of 200 km, as well as after a 24 h period of recovery. Thirty-two male ultra-distance runners participated in the study. Serum CPK showed a marked increase throughout the race and 24 h recovery period (p < 0.001). Serum CK during the race occurs mostly in the CK-MM isoform and only minutely in the CK-MB isoform and is unchanged in the CK-BB isoform. High-sensitivity C-reactive protein (hs-CRP), oestradiol, AST and ALT increased significantly from the pre-race value at 100 km and a further increase took place by the end of the 200 km run. The results of our study demonstrate a different release pattern of creatine kinase after an ultra-distance (200 km) run compared to the studies of marathon running and intense eccentric exercise, and changes in several biomarkers, indicative of muscle damage during the race, were much more pronounced during the latter half (100–200 km) of the race. However, the increases in plasma concentration of muscle enzymes may reflect not only structural damage, but also their rate of clearance.     

Nutritional lipid emulsions modulate cellular signaling and activation of human neutrophils

Although numerous studies suggest that nutritional lipids modulate human immune responses, the mechanism behind this observation remains unclear. On the basis of the hypothesis that lipids might affect cellular signaling we evaluated the effects of various lipid emulsions on two major pathways involved in neutrophil activation: second messenger (Ca(2)+) mobilization and protein kinase C (PKC) activation. Activation by opsonized yeast particles (serum-treated zymosan; STZ) increased cytosolic [Ca(2)+] ([Ca(2)+](i)) in neutrophils, with an initial slow rise that turned into a fast phase until a plateau was reached. The PKC activator 4-alpha-phorbol 12-myristate 13-acetate (PMA) markedly increased the initial STZ-induced [Ca(2)+](i) rise. This PMA effect was mimicked by emulsions containing medium-chain triglycerides (MT), but not by long-chain triglycerides (LT) or structured lipids (SL). However, like PMA, all emulsions decreased the STZ-induced [Ca(2)+](i) plateau and all activated purified PKC, suggesting that only MT emulsions activate PKC in the context of the intact cell. MT, like PMA, evoked a leftward shift of the dose-response curve for the STZ-induced [Ca(2)+](i) rise, indicating PKC-dependent sensitization of neutrophils for stimulation by STZ. This study is the first to show that nutritional lipids distinctively modulate cellular signaling and stimulation of neutrophils through effects on calcium mobilization and PKC activation: i) MT emulsions sensitize neutrophils for STZ in a PKC-dependent manner, and ii) MT, LT, and SL emulsions all reduce the stimulatory effect of STZ in a nonspecific manner. -- Wanten, G., S. van Emst-de Vries, T. Naber, and P. Willems. Nutritional lipid emulsions modulate cellular signaling and activation of human neutrophils. J. Lipid Res. 2001. 42: 428--436.     

Muscle cross-bridges bound to actin are disordered in the presence of 2,3-butanedione monoxime.

Electron paramagnetic resonance spectroscopy was used to monitor the orientation of muscle cross-bridges attached to actin in a low force and high stiffness state that may occur before force generation in the actomyosin cycle of interactions. 2,3-butanedione monoxime (BDM) has been shown to act as an uncompetitive inhibitor of the myosin ATPase that stabilizes a myosin.ADP.P(i) complex. Such a complex is thought to attach to actin at the beginning of the powerstroke. Addition of 25 mM BDM decreases tension by 90%, although stiffness remains high, 40-50% of control, showing that cross-bridges are attached to actin but generate little or no force. Active cross-bridge orientation was monitored via electron paramagnetic resonance spectroscopy of a maleimide spin probe rigidly attached to cys-707 (SH-1) on the myosin head. A new labeling procedure was used that showed improved specificity of labeling. In 25 mM BDM, the probes have an almost isotropic angular distribution, indicating that cross-bridges are highly disordered. We conclude that in the pre-powerstroke state stabilized by BDM, cross-bridges are attached to actin, generating little force, with a large portion of the catalytic domain of the myosin heads disordered.     

Thermodynamic properties of the kinesin neck-region docking to the catalytic core

Kinesin motors move on microtubules by a mechanism that involves a large, ATP-triggered conformational change in which a mechanical element called the neck linker docks onto the catalytic core, making contacts with the core throughout its length. Here, we investigate the thermodynamic properties of this conformational change using electron paramagnetic resonance (EPR) spectroscopy. We placed spin probes at several locations on the human kinesin neck linker and recorded EPR spectra in the presence of microtubules and either 5'-adenylylimidodiphosphate (AMPPNP) or ADP at temperatures of 4-30 degrees C. The free-energy change (DeltaG) associated with AMPPNP-induced docking of the neck linker onto the catalytic core is favorable but small, about 3 kJ/mol. In contrast, the favorable enthalpy change (DeltaH) and unfavorable entropy change (TDeltaS) are quite large, about 50 kJ/mol. A mutation in the neck linker, V331A/N332A, results in an unfavorable DeltaG for AMPPNP-induced zipping of the neck linker onto the core and causes motility defects. These results suggest that the kinesin neck linker folds onto the core from a more unstructured state, thereby paying a large entropic cost and gaining a large amount of enthalpy.     

Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila

Background  

We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process.     

Nutrient and drug effects on cholesterol metabolism in the laying hen

The laying hen is a highly dynamic model for studies of cholesterol metabolism. Cholesterol biosynthesis takes place primarily in the liver where it is regulated by both diet and drugs. Ovarian cholesterol biosynthesis follows a pattern different from that in liver and is not influenced by dietary fat or cholesterol. The hen responds to high levels of dietary polyunsaturated fat by increasing cholesterol biosynthesis, egg cholesterol deposition, and fecal bile acid excretion. Dietary cholesterol curtails liver cholesterol biosynthesis and may or may not result in increased egg cholesterol deposition and/or increased fecal steroid excretion depending on the level of cholesterol intake. Dietary plant sterols and fiber may moderate egg cholesterol deposition but the conditions under which this takes place are not well defined. D-Thyroxin reduces blood cholesterol, increases blood sterol turnover, and increases egg cholesterol concentration. Triparanol and azasterols prevent desmosterol conversion to cholesterol with resultant appearance of both sterols in blood and eggs. Probucol moderates egg cholesterol deposition by reducing synthesis and/or transfer of the sterol to the egg. Implications for the use of the hen in cholesterol metabolism studies are discussed.