A rapid and sensitive method for measuring monooxygenase activities in hepatocytes cultured in 96-well plates

Summary Measurement of biotransformation activities in cells is of great importance for drug metabolism and toxicologic studies. It is currently done by measuring the enzymatic activities in partially purified microsomes. In the present work we report on a rapid, easy, sensitive, and reproducible fluorimetric assay for quantifying cytochrome P450-dependent monooxygenase activities (P450IA1, P450IIB1) in hepatocytes cultured in 96-well plates. The procedure involves the direct determination of enzymatic activities in intact hepatocytes while avoiding cell homogenization, thereby permitting use of a the reduced number of cells and allowing cultured cells to be used in later experiments. Substrates (7-ethoxyresorufin, 7-pentoxyresorufin) are added to culture medium and metabolized by hepatocytes. After enzymatic deconjugation, the fluorescent resorufin present in culture medium is quantified by means of a microplate fluorimetric reader. Major advantages of this technique, as compared to other available methods, are: a) no cell disruption is required; b) activity can be measured with a very small number of cells; c) rapid processing time; and d) possibility of performing repeated assays with the same cell monolayer.     

免疫吸附治疗在肾移植致敏受者中的应用

目的：探讨蛋白A免疫吸附（immunoads Orption，IA）治疗对清除肾移植致敏受者体内特异性抗HLA抗体的疗效和安全性。方法：10例肾移植致敏（PRA〉50％）受者用彤新蛋白A免疫吸附柱行IA治疗，测定治疗前后血免疫球蛋白及PRA水平。结果：10例患者IA治疗次数为4～15次（中位数9）。所有患者IA治疗后血清总IgG水平都明显下降（P〈0．001），IgA和IgM也较治疗前显著降低（P〈0．01）。PRA8例转阴，1例〈30％，1例仍为100％。结论：对于肾移植致敏受者，IA是一种特异性高、安全有效的治疗措施。     

Comparative Efficacy of Intra-Articular Steroid Injection and Distension in Patients With Frozen Shoulder: A Systematic Review and Network Meta-Analysis

Objective

To compare the efficacy of intra-articular (IA) steroid injection and distension in patients with frozen shoulder.

Balance Control and Energetics of Powered Exoskeleton-Assisted Sit-to-Stand Movement in Individuals With Paraplegic Spinal Cord Injury

Objective

To quantify the effects of initial hip angle and angular hip velocity settings of a lower-limb wearable robotic exoskeleton (WRE) on the balance control and mechanical energy requirements in patients with paraplegic spinal cord injuries (SCIs) during WRE-assisted sit-to-stand (STS).

Safety of reassessment-and-release practice for mental health patients boarded in the emergency department

Objectives

Among emergency department (ED) mental health and substance abuse (MHSA) patients, we sought to compare mortality and healthcare utilization by ED discharge disposition and inpatient bed request status.

Total and regional myocardial blood flow in aortic regurgitation.

Total, phasic, and regional flow were studied in 12 open-chest dogs with aortic regurgitation. An adjustable catheter device was used to produce aortic regurgitation. Four differently labeled 7 to 9μ microspheres were injected into the left atrium during control, mild (5 to 25 per cent), moderate (25 to 50 per cent), and severe (50 to 80 per cent) regurgitation. Aortic regurgitation (AR) and the ratio of diastolic coronary blood flow to systolic coronary blood flow ($\text{DIAS}\text{SYS}$ RATIO) were measured from the electromagnetic flow tracings. The simultaneous left ventricular and aortic pressures were used to calculate $\text{DPTI}\text{SPTI}$ (diastolic pressure time index to systolic time index). Myocardial flow, flow to major subgroups, and endocardial/epicardial ratios were determined from radioisotope analysis of the left ventricle.Mean absolute control values and mean changes of key variables from control were:

3.

	Control	Mild AR	Mod AR	Severe AR
Heart rate (beats/min.)	163.42	?5.00	?4.831	?8.56
$\text{DPTI}\text{SPTI}$	1.18	?0.121	?0.241	?0.561
Dias/Sys ratio	4.23	?0.28	?1.641	?3.311
Myo. flow (ml./100g./min.)	99.90	?5.63	11.18	21.63
Endocardium (ml./100 g./min.)	97.27	?5.28	10.26	7.39
Epicardium (ml./100 g./min.)	100.15	?7.38	7.88	21.58
$\text{ENDO}\text{EPI}$ ratio	0.99	0.01	0.01	?0.11

1

Denotes significant change from control state (P = 0.05).

The phasic coronary blood flow results in this study are similar to those reported in chronic, intact anesthetized dogs; when the degree of aortic regurgitation increased, there was a significant decrease in diastolic coronary blood flow with an increase in systolic coronary blood flow. Not previously reported are the changes in the distribution of myocardial perfusion. Total myocardial flow increased slightly. There were minimal changes in blood flow to the endocardium which resulted in a slight decrease in the $\text{ENDO}\text{EPI}$ ratio and a decrease in the per cent of flow to the endocardium. These results indicate that, although acute aortic regurgitation produces significant changes in phasic coronary flow, there are much smaller effects on total and regional myocardial blood flow.     

Placental transfer of aspartate and its metabolites in the primate.

The placental transfer of aspartate was tested in pregnant monkeys infused maternally with sodium aspartate. In five animals infused at 100 mg/kg/hr, maternal plasma aspartate levels increased from 0.36 ± 0.19 to 80.2 ± 11.5 μmole/dl (mean ± SD). However, fetal plasma aspartate levels increased only slightly from 0.42 ± 0.31 to 0.98 ± 0.24 μmole/dl (p = 0.02). Erythrocyte aspartate levels were unchanged in both fetal and maternal circulation. In two animals infused at 200 mg/kg/hr, maternal plasma aspartate levels increased from 0.28 and 0.31 μmole/dl to values of 141 and 237 μmole/dl, respectively. This increase produced a significant (p = 0.001) increase in fetal plasma aspartate levels from 0.53 and 0.67 to 3.3 and 4.5 μmole/dl, respectively. Maternal plasma aspartate levels in two animals infused at 400 mg/kg/hr increased from 0.5 and 0.7 μmole/dl to 400 and 750 μmole/dl, respectively, at the end of the infusion. Fetal plasma aspartate levels increased from 0.21 and 0.25 μmole/dl to 60 and 92 μmole/dl, respectively. Maternal aspartate infusion at each level increased maternal, but not fetal, plasma taurine levels. The increase in maternal taurine levels was not in proportion to the dose of aspartate infused. Aspartate metabolites, glucose, and lactate were readily transferred across the placenta. The data indicate that aspartate, like glutamate but unlike most amino acids, is not concentrated toward the fetal circulation in the pregnant primate, and suggest that a barrier to aspartate transfer exists unless maternal plasma levels are grossly elevated.