sodium-dodecyl-sulfate and ubenimex

Recent studies show that mononuclear cells release a small peptide (molecular weight < 3,000) that stimulates chromaffin cell epinephrine secretion (1). The present study demonstrates that endotoxin (ETX) enhances this mononuclear cell-mediated epinephrine secretion and examines the potential mechanism for regulation of this peptide. Mononuclear cells from bovine spleen were cultured 24 h in serum-free media after which the supernatant (conditioned media, CM) was harvested and filtered to remove molecules with a molecular weight greater than 3,000. In vitro epinephrine secretion from bovine chromaffin cells was used as a test system and CM-secretion expressed as a percentage of total cell content. ETX challenge (1 microgram/mL) of mononuclear cell cultures significantly enhanced bioactivity of CM (control-CM = 11.8 +/- .7, ETX-CM = 17.7 +/- 2.8). Separation of cell populations by adherence to plastic revealed that T cell and/or B cell populations were the main source of the bioactive peptide(s) in unstimulated cell cultures (T/B cell = 12.9 +/- .7, M phi = 6.2 +/- .8). In contrast, ETX induced significant bioactive peptide release from the macrophage population (M phi = 6.2 +/- .8, ETX-M phi = 15.9 +/- 2.8). Polyacrylamide gel analysis revealed a small peptide in nonadherent cell CM that was present only in ETX-challenged macrophage cultures. Additionally, data are presented that demonstrate a correlation between CM bioactivity and protease activity in CM. Proteases secreted from mononuclear cells in response to ETX is hypothesized to cleave the bioactive peptide from a larger "parent" protein. This peptide may play a role in the elevation of plasma catecholamines observed in the setting of critical injury and illness and contribute to development of the systemic inflammatory response syndrome (SIRS) and subsequent shock states.

Topics: Animals; Cattle; Chromaffin Cells; Culture Media, Conditioned; Electrophoresis, Polyacrylamide Gel; Endotoxins; Epinephrine; Leucine; Leucyl Aminopeptidase; Leukocytes, Mononuclear; Macrophages; Peptides; Phytohemagglutinins; Protease Inhibitors; Sodium Dodecyl Sulfate

The effects of various surfactants and protease inhibitors on the nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) were examined in rats. No effects of bile salts and acids such as sodium glycocholate or taurocholic acid, amphoteric surfactants such as lauryldimethyl betaine, or anionic surfactants such as sodium lauryl sulfate on the absorption were found at a concentration of 1%. But non-ionic surfactants with hydrophile/lipophile balance (HLB) of 13 to 18 increased the total leukocyte numbers maximally by about 250% as a relative increase ratio to the control without surfactants. The increase in the plasma rhG-CSF concentration was obviously observed only in the presence of non-ionic surfactants, and in particular, the effects of Laureth-9 on the increase in total leukocyte numbers and plasma rhG-CSF concentration were maximal. In the presence of various kinds of protease inhibitors, the increasing effect of rhG-CSF on the total leukocyte numbers was not changed. Consequently, it is considered that the permeation of rhG-CSF through the nasal epithelium can be improved by non-ionic surfactants, but the effect of a protease inhibitor is smaller than that of the surfactant.

Topics: Absorption; Animals; Aprotinin; Bacitracin; Betaine; Detergents; Ethylmaleimide; Glycocholic Acid; Granulocyte Colony-Stimulating Factor; Humans; Immunoenzyme Techniques; Leucine; Leukocyte Count; Male; Nasal Mucosa; Phenylmethylsulfonyl Fluoride; Polidocanol; Polyethylene Glycols; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sodium Dodecyl Sulfate; Surface-Active Agents; Taurocholic Acid; Trypsin

Two forms of a neutral protease that catalyzed the hydrolysis of succinyl-Leu-Leu-Val-Tyr-MCA were isolated from porcine skeletal muscle cytosol by fractionation on DEAE-cellulose, hydroxyapatite and Sephadex G-100. The native enzyme had a molecular mass of above 1000 kDa. Peak A, which was eluted from hydroxyapatite at 50 mM phosphate, was activated 37-fold by the detergent, SDS, while peak B which was eluted at 150 mM phosphate, was activated only 2-fold. After dialysis against water, the B form showed restored ability to be activated by SDS (9.6-fold with 0.04% SDS). The activated peak B was extremely sensitive to divalent and monovalent cations such as Ca2+, Mg2+, Na+, K+ and NH+4 as well as protease inhibitors such as leupeptin, chymostatin and DFP. These results suggest that these proteases are generally latent in the cells and may be regulated by changes in the concentrations of cations in the cytosol. We call this new type of protease, ingensin.

Topics: Animals; Antipain; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, High Pressure Liquid; Coumarins; Cysteine Endopeptidases; Endopeptidases; Isoenzymes; Leucine; Leupeptins; Multienzyme Complexes; Muscles; Oligopeptides; Pepstatins; Proteasome Endopeptidase Complex; Sodium Dodecyl Sulfate; Swine