Rat alveolar macrophages were examined for the presence of subpopulations with different capacities for modulation of mitogen-induced lymphocyte proliferation and production of the monokine, lnterleukin-1 (IL-1). Alveolar macrophages lavaged from normal rats were separated into 5 density fractions by centrifugation through a continuous gradient of isosmotic colloidal silica (Percoll). Measurement of cell size and endogenous peroxidase suggested that the cells fractionated by density represented alveolar macrophages at different levels of cell maturation. Alveolar macrophages from each of the density tractions were cultured with whole lymph node cells and the mitogens, concanavilin-A and phytohemagglutinin. Functional heterogeneity was demonstrated among the fractionated cells with respect to suppression of lymphocyte mitogenesis. Alveolar macrophages from intermediate density tractions suppressed mito-genesis in a dose-dependent manner, whereas alveolar macrophages from both the lowest and the highest den...

Heterogeneity of immunologic function among subfractions of normal rat alveolar macrophages

Dietary levels of β-carotene and vitamin E have been associated with cancer prevention and to a lesser extent, with therapeutic enhancement of cancer treatment. We report on the cytotoxicity of β-carotene, vitamin E, and the combination of β-carotene and vitamin E in human SCC-25 squamous carcinoma cells under various environmental conditions found in solid tumor masses. β-Carotene was selectively cytotoxic toward normally oxygenated cells and was generally more cytotoxic at normal pH than at acidic pH (6.45). Vitamin E was selectively cytotoxic toward normally oxygenated cells following 6 h exposure at normal pH and was generally equally cytotoxic toward normally oxygenated and hypoxic cells under the other conditions tested. β-Carotene was an effective modulator of cisplatin (CDDP) cytotoxicity toward SCC-25 cells, whereas vitamin E was not. Both β-carotene and vitamin E were effective modulators of melphalan cytotoxicity toward SCC-25 cells. Treatment of SCC-25 cells with β-carotene (70 μm, 2h) resultec in a reduction in superoxide dismutase activity, in glutathione-S-transferase activity, and in nonprotein sulfhydryl levels in the cells. Exposure to vitamin E or to a combination of β-carotene and vitamin E increased the glutathione-S-transferase activity in SCC-25 cells by 40%–45% over the control value. Treatment with β-carotene, vitamin E, or canthaxanthin reduced the incorporation of [3H]-thymidine into SCC-25 cells but not that into normal human karotinocytes. The most marked reduction in [3H]-thymidine incorporation into SCC-25 cells occurred following treatment with the combination of β-carotene and melphalan. We hope to continue to explore the mechanisms of this effect and to study these combinations in vivo.

β-Carotene and/or vitamin E as modulators of alkylating agents in SCC-25 human squamous carcinoma cells

Leishmania amastigotes, obligatory parasites of macrophages, lodge and multiply within long-lived phagolysosomelike "parasitophorous vacuoles" (PV). The glycoprotein horseradish peroxidase (HRP) was shown, by light and electron microscopic cytochemistry, to enter the PVs of rat in vitro-derived bone marrow macrophages infected with Leishmania mexicana amazonensis. Uptake was obtained both in preinfected macrophages incubated with HRP and in macrophages pulsed with HRP, infected, and further incubated in ligand-free medium. Peroxidase positive and negative PVs could coexist in the same macrophages. Infected macrophages commonly displayed fewer labeled secondary lysosomes than noninfected cells. Lactoperoxidase (LP) was also shown, by light microscopy, to enter the PVs of rat macrophages. Uptake of HRP and of LP was blocked by mannan, supporting the mannose receptor mediated recognition of these ligands. Transfer of HRP to PVs was much less efficient in resident mouse peritoneal macrophages, even at 50 X higher ligand concentrations. Such macrophages expressed negligible mannose receptor function. The efficient mannan-inhibitable uptake of HRP by rat marrow macrophages was confirmed biochemically. Bulk HRP uptake in infected and noninfected cultures was found to be similar. Peroxidases should be useful in further studies of endocytosis by Leishmania-infected macrophages and in the development of lysosomotropic macrophage-targeted drug carriers.

Receptor-mediated entry of peroxidases into the parasitophorous vacuoles of macrophages infected with Leishmania Mexicana amazonensis.

delta-9-Tetrahydrocannabinol inhibits cell contact-dependent cytotoxicity of Bacillus Calmétte-Guérin-activated macrophages.

The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.

Ligand-mediated internalization of glucagon receptors in intact rat liver

Publisher Summary 5′-Nucleotidase hydrolyzes the phosphoester linkage in 5′-mononucleotides, liberating a nucleoside and inorganic phosphate. Most assays for 5′-nucleotidase depend upon measuring the rate of appearance of one of the two products of hydrolysis of the nucleotide substrate: either the inorganic phosphate or the nucleoside. The chapter describes an assay in which the nucleotide carries a radioactive label in its nucleoside portion and the labeled nucleoside generated by the enzymatic hydrolysis is separated from the substrate and counted in a scintillation spectrometer. It is important to establish that the activity observed is actually due to 5′-nucleotidase rather than a nonspecific phosphatase. 5′-Nucleotidase is most effective in hydrolyzing ribonucleoside 5′-monophosphates with little or no cleavage of the corresponding 5′-deoxyribonucleotides or 5′-ribonucleoside di- or triphosphates observed. A nucleotide analog, α, β-methylene adenosine 5′-diphosphate (AOPCP), has been reported to be a very powerful inhibitor of 5′-nucleotidase, though its efficacy may vary among different species.

5′-nucleotidase assay

Publisher Summary The Lowry and Bradford assays for protein determination provide relatively simple methods for calculating specific activities by standardizing enzyme activities to protein content. Both assays are colorimetric. The Lowry assay depends on the complexing of copper with tyrosine and tryptophan residues, whereas the Bradford method measures the binding of Coomassie Brilliant Blue G-250 to protein. The protocols are modified specifically for protein determination in Triton lysates but may be altered for different conditions by simply replacing Triton with the appropriate agent. The standards may be plotted on a linear graph of concentration versus absorbance, and unknown concentrations may then be read directly from the graph. Alternatively, a straight line may be fit to the standard data by the method of least squares and the formula generated may be used to calculate unknown concentrations. This latter method is especially convenient for use with a hand-held programmable calculator. The blank cuvette should be rinsed approximately every 10 min to avoid progressive staining of the cuvette.

Robert A. Duncan

Papers

5′-nucleotidase assay

Lowry and bradford assays for protein