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.     

Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage.

Adipose tissue represents an accessible source of mesenchymal stem cells (ADSCs), with similar characteristics to bone marrow-derived stem cells. The aim of this work was to investigate the transdifferentiation of ADSCs into hepatic lineage cells in vitro. ADSCs were obtained from human adipose tissue from lipectomy. Cells were grown in medium containing 15% AB human serum. Cultures were serum deprived for two days and exposed to a two-step protocol with two different media using growth factors and cytokines. Hepatic differentiation was assessed by RT-PCR of liver-marker genes. ADSCs exhibited a fibroblastic morphology that changed to a cuboidal shape when cells differentiated. Expression of liver genes increased when using one of the two studied media consisting of DMEM supplemented with HGF, bFGF and nicotinamide for 14 days. The results indicate that, under certain specific inducing conditions, ADSCs can be induced to differentiate into hepatic lineage in vitro. Adipose tissue may be an ideal source of high amounts of autologous stem cells.     

Alterations of motor activity circadian rhythm in rats with adjuvant arthritis

Adjuvant arthritis in rats produces alterations in the motor activity circadian rhythm. Specifically arthritic animals show a decrease in the total daily motor activity and an advance in the acrophase of the rhythm. Slight changes are also observed in the power content of the circadian harmonic as well as in the amplitude.     

The Changing Faces of the Nutcracker Esophagus

Although the nutcracker esophagus, characterized by high amplitude peristaltic contractions with mean distal amplitude greater than 180 mm Hg, is the most common esophageal motility disorder associated with noncardiac chest pain, little is known about its natural history. Therefore, we reviewed the manometric tracings of 23 patients with the nutcracker esophagus who had an average of 4.6 studies during a mean period of 32 months. Ten age-matched volunteers with normal baseline manometry who had undergone multiple studies (mean 5.8) over a mean time span of 32 months served as controls. In the 17 nutcracker patients with three or more motility studies, the variability of mean distal amplitudes between studies was 41.9% +/- 4.1 (+/- SE) compared to 27.0% +/- 3.3 for the control subjects (p less than 0.01). Highest distal pressures were noted during the first study in 11 of 17 patients (65%) compared to two of 10 controls (20%). The consistency of the diagnosis of nutcracker esophagus varied considerably: four patients always had high amplitude pressures, three patients only had the nutcracker diagnosis on the initial study, and 10 patients intermittently had pressures in the nutcracker range. Overall, these 17 patients had the diagnosis of the nutcracker esophagus confirmed on only 54% of subsequent studies. Changes in motility patterns were intermittently seen in six of 23 patients: one diffuse spasm and five nonspecific motility disorders. None of the control subjects developed high amplitude contractions or changed their motility pattern on serial testing. The possible pathophysiological implications of the changing faces of the nutcracker esophagus are discussed.