Modulation of recombinant human prostate-specific antigen: activation by Hofmeister salts and inhibition by azapeptides. Appendix: thermodynamic interpretation of the activation by concentrated salts.

Prostate specific antigen (PSA, also known as human kallikrein 3) is an important diagnostic indicator of prostatic disease. PSA exhibits low protease activity (>10(4)-fold less than chymotrypsin) under the usual in vitro assay conditions. In addition, PSA does not react readily with prototypical serine protease inactivators. We expressed human PSA (rh-PSA) in Escherichia coli and have demonstrated that rh-PSA has properties similar to those of native PSA isolated from human seminal fluid. Both PSA and rh-PSA are >10(3)-fold more active in the presence of 1.3 M Na(2)SO(4). This activation is anion-dependent, following the Hofmeister series when normality is considered: SO(4)(2)(-) approximately citrate > Ac(-) > Cl(-) > Br(-) > I(-). The nature of the cation has little effect on salt activation. The rate of inactivation of rh-PSA by DFP is 30-fold faster in the presence of 0.9 M Na(2)SO(4), and the rate of inactivation by Suc-Ala-Ala-Pro-Phe-CK is >20-fold faster under these conditions. Azapeptides containing Phe or Tyr at position P(1) also inactivate rh-PSA in the presence of high salt concentrations. These compounds represent the first described inhibitors designed to utilize the substrate binding subsites of PSA. CD spectroscopy demonstrates that the conformation of rh-PSA changes in the presence of high salt concentrations. Analytical ultracentifugation and dynamic light scattering indicate that PSA remains monomeric under high-salt conditions. Interestingly, human prostatic fluid contains as much as 150 micro mol citrate/g wet weight, which suggests that salt concentrations may regulate PSA activity in vivo.

Dietary marine lipids suppress continuous expression of interleukin-1 beta gene transcription.

n-3 Polyunsaturated fatty acids abundant in marine lipids suppress certain inflammatory and immune reactions, and dietary marine lipid supplements have antiinflammatory effects in experimental and human autoimmune disease. Previous work by other investigators demonstrated that dietary marine lipid supplements suppressed production of cytokines from stimulated human peripheral blood mononuclear cells ex vivo. The present study further documents the ability of n-3 fatty acids to inhibit cytokine formation, and in part defines the mechanism of the inhibition of production of interleukin-1 beta (IL-1 beta) by dietary n-3 fatty acid. Female BALB/c mice were each fed a fat-free balanced diet to which was added either a refined fish oil (FO) preparation as a source of n-3 fatty acid, or beef tallow (BT), which consisted primarily of saturated and monoenoic fatty acids. After ingesting the experimental diets for periods ranging from 3 to 12 wk. spleen cell preparations were stimulated ex vivo with either lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and proIL-1 beta mRNA (IL-1 beta mRNA) was measured by northern analysis. Levels of IL-1 beta mRNA in both LPS- and PMA-stimulated cells from BT-fed mice were elevated to a greater extent than in cells from FO-fed mice, at most concentrations of LPS and PMA. Stability of LPS-stimulated mRNA levels after actinomycin D was similar for BT and FO groups, indicating that lower levels of IL-1 mRNA with FO groups was related to suppressed IL-1 gene transcription and not due to accelerated transcript degradation. Nuclear run-on transcription assays revealed a more transient expression of the IL-1 beta gene in LPS-stimulated spleen cells from FO-fed mice compared to cells from BT-fed mice. We conclude that dietary marine lipids reduce transient expression of the IL-1 beta gene in stimulated splenic monocytic cells. Preliminary results from nuclear run-on transcription assays indicate that n-3 fatty acids may not change the initial rate of gene transcription but may promote more rapid shutting down of transcription of this gene after induction than do alternative lipids.

Modification of spleen phospholipid fatty acid composition by dietary fish oil and by n-3 fatty acid ethyl esters.

We have compared the effects of diets containing purified ethyl esters of either eicosapentaenoic acid, docosahexaenoic acid, or a mixture of both of these compounds, with diets containing either purified fish oil or beef tallow on spleen phospholipid fatty acid composition. Autoimmune mice, the (NZB x NZW)F1 strain, were fed with experimental diets for 14 weeks, after which spleen phospholipids were extracted and separated into classes by HPLC, and the alkenylacyl, alkylacyl, and diacyl subclasses of glycerylphosphatidylethanolamine and glycerylphosphatidylcholine were resolved as their benzoyl esters by HPLC. Fatty acids were analyzed by capillary gas-liquid chromatography of their methyl esters. Each of the marine lipid diets suppressed n-6 fatty acids and elevated n-3 fatty acids in all phospholipids. The eicosapentaenoic acid ethyl ester diets led to high levels of eicosapentaenoic acid and docosapentaenoic acid (C22:5n-3), but little or no increase in docosahexaenoic acid. The docosahexaenoic acid ethyl ester diets elevated docosahexaenoic acid, docosapentaenoic acid, and eicosapentaenoic acid in all phospholipids, indicating that extensive retroconversion of 22 carbon n-3 fatty acids had occurred. These results document changes in the fatty acid composition of mammalian phospholipids that are induced by dietary fish oil triglycerides and by dietary long chain n-3 fatty acid ethyl esters.

Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils.

Earlier studies demonstrated that dietary omega-3 polyunsaturated fatty acid (PUFA) supplementation attenuates the chemotactic response of neutrophils and the generation of leukotriene (LT) B4 by neutrophils stimulated with calcium ionophore; however, the mechanisms and relationship of these effects were not examined. Neutrophils and monocytes from eight healthy individuals were examined before and after 3 and 10 wk of dietary supplementation with 20 g SuperEPA daily, which provides 9.4 g eicosapentaenoic acid (EPA) and 5 g docosahexaenoic acid. The maximal neutrophil chemotactic response to LTB4, assessed in Boyden microchambers, decreased by 69% after 3 wk and by 93% after 10 wk from prediet values. The formation of [3H]inositol tris-phosphate (IP3) by [3H]inositol-labeled neutrophils stimulated by LTB4 decreased by 71% after 3 wk (0.033 +/- 0.013% [3H] release, mean +/- SEM) and by 90% after 10 wk (0.011 +/- 0.011%) from predict values (0.114 +/- 0.030%) as quantitated by beta-scintillation counting after resolution on HPLC. LTB4-stimulated neutrophil chemotaxis and IP3 formation correlated significantly (P < 0.0001); each response correlated closely and negatively with the EPA content of the neutrophil phosphatidylinositol (PI) pool (P = 0.0003 and P = 0.0005, respectively). Neither the affinities and densities of the high and low affinity LTB4 receptors on neutrophils nor LTB4-mediated diglyceride formation changed appreciably during the study. Similar results were observed in neutrophils activated with platelet-activating factor (PAF). The summed formation of LTB4 plus LTB5 was selectively inhibited in calcium ionophore-stimulated neutrophils and was also inhibited in zymosan-stimulated neutrophils. The inhibition of the summed formation of LTB4 plus LTB5 in calcium ionophore-stimulated neutrophils and in zymosan-stimulated neutrophils did not correlate significantly with the EPA content of the PI pool. The data indicate that dietary omega-3 PUFA supplementation inhibits the autoamplification of the neutrophil inflammatory response by decreasing LTB4 formation through the inactivation of the LTA epoxide hydrolase and independently by inhibiting LTB4- (and PAF) stimulated chemotaxis by attenuating the formation of IP3 by the PI-selective phospholipase C. This is the initial demonstration that dietary omega-3 PUFA supplementation can suppress signal transduction at the level of the PI-specific phospholipase C in humans.

Dietary marine lipids suppress murine autoimmune disease.

Dietary marine lipids reduce both mortality and the severity of glomerulonephritis in inbred murine strains which develop spontaneous autoimmune disease. The protective effects of marine lipids appear to be accounted for by the major n-3 fatty acids in these preparations, 20:5 and 22:6. The n-3 fatty acids in dietary fish oil are extensively incorporated into several lipid classes in the spleen of autoimmune mice, including phosphatidylinositol, phosphatidylethanolamine, plasmalogens and saturated ether-linked phospholipids as well as diacylphosphoglycerides. The effects of dietary marine lipids on autoimmune disease in experimental models are highly specific. Careful controlled trials will be required to establish the role of dietary marine lipids in the therapy of human autoimmune disease.