Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.     

Time-dependent measurement of differential digital plethysmogram in bicycle ergometer exercise

Time-dependent measurements of differential digital plethysmogram during exercise were made on five male subjects. The results obtained were as follows; Differential digital plethysmogram (delta DPG) was obtained by using biophysical amplifier with the time constant of 0.03 seconds which minimized the fluctuation of the baseline in digital plethysmogram (DPG). A linear relationship was shown in P wave amplitude of both delta DPG and DPG. The decrease in delta DPG-P waves was observed in visual concentrations, mental learning and arithmetic, initial inspiratory phase with tachycardia, maximal inspiratory and/or expiratory breath holding, and head-up tilt at 60 degrees or over. The increase in delta DPG-P waves was obtained at the expiratory phase with bradycardia and in the effect of alcohol intake. During 15 minutes of bicycle ergometer exercise at 750 kpm/min, the P wave amplitude in delta DPG decreased to 77% of the control in the first one minute of exercise, and then gradually increased to 218% at the final stage of exercise (p less than 0.01). Heart rate measured simultaneously increased, as compared from the beginning of exercise. P wave amplitude and heart rate after exercise decreased progressively to the control level. It is suggested that the initial decrease in P wave amplitude of delta DPG couples with the dominant activity of the sympathetic vasoconstrictor, and the final increase in P waves is due to the compound factors of the increased cardiac output and arteriolar vasodilation.     

An Epidermal Growth Factor Motif from Del1 Protein Increases the Efficiency of In Vivo Gene Transfer with a Non-Viral Vector

Increasing the efficiency of gene transfer using non-viral vectors, which have the potential to be safe and economical, would improve upon available options for gene therapy. We previously reported that the third EGF motif of the extracellular matrix protein Del1 (E3) increases the transfection efficiency of non-viral vector methods. Here, we asked if E3 could increase the in vivo transfection efficiency of a polyplex-based approach. To test this, cDNA encoding a heat-stable alkaline phosphatase (AP) was first injected intravenously into mice along with recombinant E3. After 24 h, exogenous AP activity in serum was measured. We found that the introduction of E3 resulted in 50 % more AP activity as compared to the control. We next tested transfection into a tumour explant of SCCKN cells, an oral carcinoma-derived cell line. To do this, a cDNA encoding yellow fluorescent protein was locally injected into a tumour explant, followed by local injection of recombinant E3. Use of E3 increased the number of transfected cells to 2.5 times that of the control. Histochemical staining revealed that E3-induced apoptosis in a tumour explant. The data suggest that E3 might be a useful tool for cancer gene therapy using non-viral vectors.     

The recovery of slow axonal transport after a single intraperitoneal injection of beta, beta'-iminodipropionitrile in the rat

A single intraperitoneal injection of beta, beta'-iminodipropionitrile (IDPN) at a dose of 1.5 g/kg was given to 4-week-old rats. Immediately following, or at 1, 2, 3, 5, 10, and 15 weeks after IDPN injection, [35S]methionine was introduced into the anterior horn area of the lumbar cord. Labeled axonal proteins in the sciatic nerve were analyzed electrophoretically and fluorographically at 5, 10, and 15 weeks post-labeling. Labeled neurofilament proteins halt for a short period just after IDPN injection, then continue migrating distally, though at a slower rate, and finally the transport of affected neurofilament proteins completely recovers by 6 weeks post-labeling.     

Changes in Organization and Axonal Transport of Cytoskeletal Proteins During Regeneration

Changes in solubility and transport rate of cytoskeletal proteins during regeneration were studied in the motor fibers of the rat sciatic nerve. Nerves were injured by freezing at the midthigh level either 1-2 weeks before (experiment I) or 1 week after radioactive labeling of the spinal cord with L-[35S]methionine (experiment II). Labeled proteins in 6-mm consecutive segments of the nerve 2 weeks after labeling were analyzed following fractionation into soluble and insoluble populations with 1% Triton at 4 degrees C. When axonal transport of newly synthesized cytoskeleton was examined in the regenerating nerve in experiment I, a new faster component enriched in soluble tubulin and actin was observed that was not present in the control nerve. The rate of the slower main component containing most of the insoluble tubulin and actin together with neurofilament proteins was not affected. A smaller but significant peak of radioactivity enriched in soluble tubulin and actin was also detected ahead of the main peak when the response of the preexisting cytoskeleton was examined in experiment II. It is thus concluded that during regeneration changes in the organization take place in both the newly synthesized and the preexisting axonal cytoskeleton, resulting in a selective acceleration in rate of transport of soluble tubulin and actin.