Role of the propeptide and gamma-glutamic acid domain of factor IX for in vitro carboxylation by the vitamin K-dependent carboxylase

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the processive carboxylation of specific glutamates in a number of proteins related to blood coagulation and bone. To address the independent importance of the propeptide, gamma-carboxyglutamic acid (Gla) domain and elements beyond the Gla domain of factor IX in vitamin K-dependent carboxylation, we have examined the kinetics of carboxylation of peptides containing (1) propeptide and Gla domain, (2) the Gla domain alone, (3) uncarboxylated bone Gla protein, (4) propeptide followed by the entire uncarboxylated factor IX molecule, and (5) the factor IX propeptide followed by a non-Gla domain sequence. Our studies indicate that peptides with a covalently linked propeptide have Km values similar to the physiological substrate of the carboxylase. In contrast, the Gla domain of factor IX has a >/=230-fold higher Km for the carboxylase than the corresponding peptide with a covalently linked propeptide. This contrasts with bone Gla protein, another vitamin K-dependent protein, which appears not to require a covalently linked propeptide for high-affinity binding to the carboxylase. Analysis of the carboxylation products of a propeptide/non-Gla domain substrate indicate that it is carboxylated multiple times in a processive manner. These studies show that the perceived binding affinity of the carboxylase substrate and processivity is conferred by the propeptide without requiring the conserved Gla domain sequences and that factor IX and bone Gla protein may have distinct mechanisms of interacting with the carboxylase.     

Comparison of four methods for forage nitrate analysis

Twenty forage samples were collected and selected for variation in nitrate content. Each forage samples was analyzed 4 times by 4 different methods: diphenylamine spot plate, spectrophotometric, nitrate-selective electrode, and high-performance liquid chromatographic. Five feed extracts were spiked with 2 different amounts of nitrate and analyzed by each method. The spectrophotometric and nitrate-selective electrode had similar percent recoveries, which were close to 100%. The nitrate-selective electrode method had the least variation of the 4 methods. The diphenylamine spot plate method had the poorest average recovery, greatest variation, and was the least accurate. The average coefficients of variation for all samples within a method were 15%, 12%, 6.4%, and 16 for the diphenylamine spot plate, spectrophotometric, nitrate-selective electrode, and high-performance liquid chromatographic methods, respectively. The variation in the nitrate-selective electrode method was lower (P < 0.05) than the other methods. The results from this study suggest that the nitrate-selective electrode method is more accurate and precise than the other methods of analysis tested.     

Comparison of the effects of class I anti-arrhythmic drugs, cibenzoline, mexiletine and flecainide, on the delayed rectifier K+ current of guinea-pig ventricular myocytes

The effect of class I anti-arrhythmic drugs, cibenzoline, mexiletine and flecainide, on the delayed rectifier potassium current (IK) in guinea-pig ventricular myocytes was studied using whole cell voltage clamp techniques and under blockade of the L-type calcium current by 5 microM nitrendipine. IK consisted of two different current systems, as reported by Sanguinetti and Jurkiewicz (1990), i.e. an E4031-sensitive rapidly activating component (IKr) with a strong inward-going rectification property and an E4031-insensitive slowly activating component (IKs) with little rectification. Cibenzoline (30 microM) decreased both IKr and IKs while flecainide (10 and 30 microM) decreased the IKr exclusively. Mexiletine (30 microM), in contrast, affected neither IKr nor IKs. Since the inhibition of IK(r) and/or IKs prolongs duration of action potentials and refractory periods, class I drugs which also possess the class III effect may have additional effects in treating certain re-entrant arrhythmias.     

中厚板淬火过程的横向冷却曲线

针对中原板淬火过程中均匀喷水时造成钢板变形的现象，提出了非均匀喷水的补偿方法，采用有限元分析方法，对钢板在多股集管冲击射流作用下的温度场分布进行了数值模拟，得到钢板横向冷却曲线，为无约束淬火机上集管的工艺设计提供了理论依据。     

Sensitive fused-silica capillary gas chromatographic assay using electron-capture detection for indomethacin in ovine fetal fluids

A sensitive gas chromatographic (GC) method with electron-capture detection (ECD) has been developed to quantitate indomethacin (IND) in plasma, urine, amniotic, and tracheal fluids obtained from the pregnant sheep model. IND and the internal standard, alpha-methylindomethacin (alpha-Me-IND) are extracted by a simple liquid-liquid extraction procedure using ethyl acetate and derivatized with N-methyl-N-(tert.-butyldimethyl-silyl)trifluoroacetamide (MTBSTFA) at 60 degrees C for 50 min. The limit of quantitation (LOQ) is 1 ng/ml with a C.V. < 10% and signal-to-noise ratio > 10. Recoveries from all fluids were greater than 80%. Calibration curves were linear over the range of 1-32 ng/ml with a coefficient of determination (r2) > 0.999. Inter- and intra-day coefficients of variation were < 10% at concentrations of 2-32 ng/ml, and < 20% at the LOQ. Applicability of the developed method is demonstrated for a pharmacokinetic study of IND samples collected following long-term infusion of IND in a chronically instrumented ovine fetus.     

Accuracy and variability of leg velocities during concentric and eccentric actions of the quadriceps femoris muscles

This study examined the ability to control leg velocities during concentric and eccentric actions of the right quadriceps muscles. Ten healthy women (M age = 25.9 +/- 3.5 yr.) were tested using the Isotonic Program of the KIN-COM II 500H dynamometer. They attempted to match velocity tracings of 10 degrees, 20 degrees, and 40 degrees/sec. through 70 degrees of knee range of motion at a load equal to 10% of their maximal mean concentric force. The actual mean velocities, mean percent deviation from the target velocities, and the coefficient of variation for both actions were calculated for 15 degrees-75 degrees (full range of motion), 15 degrees-45 degrees (shorter range of motion), and 46 degrees-75 degrees (longer range of motion). Separate one-way analyses of variance with two trial factors (action x velocity) showed faster concentric velocities through the full and longer ranges of motion, and faster eccentric velocities through the shorter range of motion. Mean percent deviations indicated that the eccentric velocities were generally more accurate within all ranges of motion. Larger concentric coefficients of variation were found within the full and longer ranges of motion, and the coefficients of variation for both actions decreased as the velocities increased. An exaggerated 'velocity overshoot' at the onset of both actions probably contributed to differences in the velocities and coefficients of variation. The results indicated differences between the concentric and eccentric actions, explained in part by the testing methodology used and by the known mechanical and physiological characteristics of the two muscle actions.