Синдром хронічної втоми: взаємозв'язок вправ та імунної дисфункції

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CA), aprotinin, leupeptin, pefabloc-SC, and EDTA (Roche Biochemicals, Mannheim, Germany). Protease inhibitors are required for preventing proteolytic cleavage. Standard laboratory procedures were used to separate serum from coagulated blood, and to store it at -70°C until analysis. A modified Bradford assay method (Bio-Rad Laboratories, Hercules, CA) was used for quantification of total proteins in the patients' cell extracts. The probe specifically attaches to 2'-5'A binding proteins like 80 kDa RNase L and 37 kDa RNase L. Two hundred milligrams of PBMC extract was incubated with 2'-5' A trimer radiolabeled at the 3' end with 32P-pCp, at 2-4°C for 15 min. In addition, it was covalently attached to the binding proteins by the addition of cyanoborohydride (20 raM in 100 rnM of phosphate buffer, pH 8. 0). This reduction reaction was allowed to progress for 20 min at 2-4°C. A tracking dye were added to the samples, and incubated at 95CC for 5 min followed by separation using standard SDS-PAGE with a 4% stacking and a 10% separating gel. The gel was dried and autoradiography was used to detect the radioactivity of the marked probe (Bio-Rad Laboratories Molecular Imager® Fx, Hercules, CA). Densitometric analysis of the autoradiographs was followed by quantification of any present 2'-5' A binding proteins (using specializes software: Quantity One® Software, Bio-Rad Laboratories, Hercules, CA). For ratio of the 37 kDa RNase L isoform over the 83 kDa RNase L, a threshold value of 0. 4 for the diagnosis of CFS was found to have a sensitivity of 91% and a specificity of 71% (29). Using a threshold value of 0. 5, the RNase L-ratio was able to distinguish between CFS patients (abnormal in 41 of 57 subjects or 72%), healthy controls (3/28 or 11%), and patients with Fibromyalgia- (0/11) and Depression (0/14) (3). In another study the amount of 37 kDa RNase L was able to distinguish between CFS patients (/V = 53) and healthy controls (N = 26; P = 0. 007) (27).

The RNase L enzymatic activity (enzymatic assay) was assessed as described previously (24). In several studies, the RNase L activity was able to distinguish between CFS patients and healthy controls, with higher activity in the patients' group (27, 28). For the PKR activity measurement, PAGE-separated proteins were transferred to a membrane and visualized by immunodetection. In detail, the membranes were prepared by cutting out membranes of appropriate size (15 X 10 cm) and the membranes were prewet in Methanol 100%, shaken gently until soaked, and then the liquid was poured off. The membranes were immersed in Towbin buffer, and were shaken gently (for a minimum of 2-3 min) until soaked. To remove the gel, the braces from the glass plates were removed from the slab gel unit from the electrophoresis system, the spacers were removed from between the glass plates using a spatula and opened up, the upper glass plate was lifted up, leaving the gel on the bottom glass plate, and the gel was adhered to the membrane. Next, the Mylar mask was placed on the bottom of the Electroblot instrument. For each gel/membrane, 2 X 4 pieces of Whatmann were cut out and soaked in Towbin buffer. To make a «sandwich, « four layers of Whatmann, PVDF- or nitrocellulose membrane, Poly Acrylamide Gel, and four layers of Whatmann were placed on top of the Mylar mask. Air bubbles were avoided, and equal contact was ascertained by rolling smoothly over the surfaces with a plastic pipette.

For the electroblot, the lid was put on the electroblot instrument, the electrodes were connected to the power supply, a weight was put on the lid (less than 1 kg to avoid buffer being squeezed out of the sandwich), and a current of 0. 8 mA • cm»2 was applied for 2 h (i. e., 240 mA for two membranes of 150 cm2 each). Expose the blotted membranes to air at room temperature (RT) for at least 1 h to make sure the blotted membrane (s) is (are) dry. To visualize the proteins, the total surface of blotted membranes (regular membrane = 10 X 15 cm = 150 cm2) was calculated and the membranes were prewet by soaking them in Methanol 70% (approximately 12 mL per blot), shaking until they were completely wet, and removing the Methanol by gently pouring it off. The membranes were washed two times with phosphate-buffered saline (PBS) -Tween® (5 min each at room temperature while gently shaking -0. 25 mL-cm-2 = 37. 5 mL for regular membrane). Next, the membranes were blocked with 5% NF milk in PBS-Tween® for 1 h at room temperature while gently shaking or overnight at 4°C, and the tray was covered with a lid or with aluminum foil (0. 25 mL-cm-2). For incubation with the primary antibody, a dilution (dependent on the manufacturer's instructions)