Protein and other compositional differences of the extracellular material from slimy and non-slimy colonies of non-mucoid Pseudomonas aeruginosa

The composition of extracellular material produced by non-mucoid strains of Pseudomonas aeruginosa differed when grown on surfaces that either did or did not induce the formation of slime under conditions where the medium was identical. The nature of the changes in protein composition indicated that protein expression differed in the course of growth on the two surfaces, and hence that there were physiological consequences associated with growth under conditions which do or do not lead to slime formation. The compositional differences also included elevated levels in extracellular material from the slimy colonies of two virulence factors, protease and rhamnolipids.     

Malignant ascites of serous papillary ovarian adenocarcinoma. An immunocytochemical study of the tumor cells

In 17 malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary, the reaction patterns of the tumor cells to monoclonal antibodies (MAbs) against surface antigens were studied and compared with the reaction patterns of mesothelial cells in the same effusions. The following surface markers were used with the adhesive slide method: epithelial membrane antigen (EMA), human epithelium-specific cell surface antigen (HEA-125), human endothelial antigen (BMA-120), carcinoembryonic antigen (CEA 3-13), an antibody against natural killer cells and cytotoxic cells (BMA-070), granulocyte antigen (Leu M1) and leukocyte antigen of class I (HLA-1). In all cases, from 30% to 95% of the tumor cells reacted with EMA and HEA-125. Tumor cells showed a positive staining with CEA 3-13 in only five cases. In all cases, from 75% to 95% of the tumor cells reacted positively with BMA-120. The reactivity of a few mesothelial cells with EMA and of all mesothelial cells with BMA-120 did not interfere with the identification of positive tumor cells since the reaction patterns were different. Interestingly, our study demonstrated that BMA-070, an MAb identifying natural killer cells and cytotoxic cells, is also a most useful tumor marker. The same was found to be true for Leu M1, an MAb originally thought to react only with granulocytes. The tumor cells showed a partial or total loss of the expression of HLA-1 reactivity. Since all cases were immunocytochemically positive for tumor cells while conventional cytology was positive in only 13 of the cases, the immunocytochemical analysis of malignant peritoneal effusions due to papillary serous adenocarcinoma of the ovary seems able to improve the cytologic diagnosis of the fluids.     

Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Stimulation of receptor-dependent and receptor-independent pathways of low-density lipoprotein degradation in arterial smooth muscle cells by platelet-derived growth factor

Platelet-derived growth factor (PDGF), a powerful mitogen released by platelets, promoted the degradation of low-density lipoprotein (LDL) by cultured primate arterial smooth muscle cells and human skin fibroblasts by stimulating both receptor-mediated and LDL-receptor-independent uptake of LDL. Stimulation of LDL-receptor-independent LDL uptake and degradation by PDGF was demonstrated in three ways. First, the small amount of LDL that was degraded by LDL-receptor-negative skin fibroblasts was stimulated by PDGF. Second, PDGF led to increased degradation of LDL that had been reductively methylated to prevent its binding to LDL receptors. Third, 125I-labeled LDL degradation was stimulated by PDGF in the presence of high concentrations of unlabeled LDL, i.e., conditions under which the contribution of the LDL receptor to cellular uptake and degradation is reduced. These observations suggest that mitogens, as typified by PDGF, can facilitate the cellular delivery of LDL cholesterol by both LDL-receptor-mediated and non-LDL-receptor-mediated mechanisms to provide exogenous cholesterol for use during cell replication.     

Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein

We have investigated the esterification by liver membranes of retinol bound to cellular retinol-binding protein (CRBP). When CRBP carrying [3H]retinol as its ligand was purified from rat liver cytosol and incubated with rat liver microsomes, a significant fraction of the [3H]retinol was converted to [3H]retinyl ester. Esterification of the CRBP-bound [3H]retinol, which was maximal at pH 6-7, did not require the addition of an exogenous fatty acyl group. Indeed, when additional palmitoyl-CoA or coenzyme A was provided, the rate of esterification increased either very slightly or not at all. The esterification reaction had a Km for [3H]retinol-CRBP of 4 +/- 0.6 microM and a maximum velocity of 145 +/- 52 pmol/min/mg of microsomal protein (n = 4). The major products were retinyl palmitate/oleate and retinyl stearate in a ratio of approximately 2 to 1 over a range of [3H]retinol-CRBP concentrations from 1 to 8 microM. The addition of progesterone, a known inhibitor of the acyl-CoA:retinol acyltransferase reaction, consistently increased the rate of retinyl ester formation when [3H]retinol was delivered bound to CRBP. These experiments indicate that retinol presented to liver microsomal membranes by CRBP can be converted to retinyl ester and that this process, in contrast to the esterification of dispersed retinol, is independent of the addition of an activated fatty acid and produces a pattern of retinyl ester species similar to that observed in intact liver. A possible role of phospholipids as endogenous acyl donors in the esterification of retinol bound to CRBP is supported by our observations that depletion of microsomal phospholipid with phospholipase A2 prior to addition of retinol-CRBP decreased the retinol-esterifying activity almost 50%. Conversely, incubating microsomes with a lipid-generating system containing choline, CDP-choline, glycerol 3-phosphate, and an acyl-CoA-generating system prior to addition of retinol-CRBP increased retinol esterification significantly as compared to buffer-treated controls.     

Time-resolved fluorescence and 1H NMR studies of tyrosyl residues in oxytocin and small peptides: correlation of NMR-determined conformations of tyrosyl residues and fluorescence decay kinetics

Steady-state and time-resolved fluorescence properties of the single tyrosyl residue in oxytocin and two oxytocin derivatives at pH 3 are presented. The decay kinetics of the tyrosyl residue are complex for each compound. By use of a linked-function analysis, the fluorescence kinetics can be explained by a ground-state rotamer model. The linked function assumes that the preexponential weighting factors (amplitudes) of the fluorescence decay constants have the same relative relationship as the 1H NMR determined phenol side-chain rotamer populations. According to this model, the static quenching of the oxytocin fluorescence can be attributed to an interaction between one specific rotamer population of the tyrosine ring and the internal disulfide bridge.     

Steroid-binding site of human and rabbit sex steroid binding protein of plasma: fluorescence characterization with equilenin

The interaction of the estrogen d-3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one (equilenin) with the human and rabbit sex steroid binding proteins (hSBP and rSBP, respectively) has been investigated by using fluorescence and absorption spectroscopy. Equilenin competes for the binding of 5 alpha-dihydrotestosterone. The calculated binding constant of equilenin for rSBP is 1.9 X 10(7) M-1 at 4 degrees C, which can be compared with the binding constant of 5.7 X 10(7) M-1 reported for hSBP [Ross, J.B.A., Torres, R., & Petra, P.H. (1982) FEBS Lett. 149, 240]. The results of fluorescence quenching experiments with the collisional quenchers KI and acrylamide indicate that the bound steroid has limited accessibility to the bulk solvent and that there are no anionic surface groups near the steroid-binding site. The fluorescence excitation spectra of SBP-equilenin complexes are similar to the absorption spectra of equilenin in low-dielectric solvents. The fluorescence emission of the SBP-equilenin complexes, however, exhibits wavelength shifts (red shifts) opposite to those of the steroid in low-dielectric solvents or complexed with beta-cyclodextrin (blue shifts) but similar to the red shift produced by addition of the proton acceptor triethylamine to equilenin in cyclohexane. These data indicate that the steroid-binding site of hSBP and rSBP is a nonpolar cavity containing a proton acceptor that participates in a specific interaction, possibly a hydrogen bond, with the 3'-hydroxyl group of the bound steroid.     

Sulfhydryl groups are essential for organic anion exchange in canine renal brush-border membranes

The effect of several sulfhydryl-modifying reagents (HgCl2, p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide) on the renal organic anion exchanger was studied. The transport of p-amino[3H]hippurate, a prototypic organic anion, was examined employing brush-border membrane vesicles isolated from the outer cortex of canine kidneys. HgCl2, PCMBS and N-ethylmaleimide inactivated p-aminohippurate transport with IC50 values of 38, 78 and 190 microM. The rate of p-aminohippurate inactivation by N-ethylmaleimide followed apparent pseudo-first-order reaction kinetics. A replot of the data gave a linear relationship between the apparent rate constants and the N-ethylmaleimide concentration with a slope of 0.8. The data are consistent with a simple bimolecular reaction mechanism and imply that one molecule of N-ethylmaleimide inactivates one essential sulfhydryl group per active transport unit. Substrate (1 mM p-aminohippurate) affected the rate of the N-ethylmaleimide (1.3 mM) inactivation: the t1/2 values for inactivation in the presence and absence of p-aminohippurate were 7.4 and 3.7 min, respectively. The results demonstrate that there are essential sulfhydryl groups for organic anion transport in the brush-border membrane. Moreover, the ability of substrate to alter sulfhydryl reactivity suggests that the latter may play a dynamic role in the transport process.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

Isolation and characterization of a carcinoma-associated antigen

GA733 is a murine IgG2a monoclonal antibody (MAb) against human gastric carcinoma and is highly tumoricidal in nude mice. The GA733 antigen is a cell surface protein with two subunits of 30,000 and 40,000 daltons. The antigen isolated by immunoaffinity chromatography consists mainly of the 30,000-dalton subunit which bears the GA733 epitope. This subunit displayed several isoelectric points between 6.9 and 7.7. Anti-colon carcinoma MAb 17-1A also detects this antigen, but probably binds to a different epitope.