INTRODUCTIONBone marrow-derived macrophages (BMM) are primary macrophage cells, derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. The efficiency of the differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, the BMMs are suitable for numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages have a unique ability to ingest microbes. We describe a test for the phagocytic efficiency of BMMs by exposing them to fluorescently labeled yeast zymosan bioparticles. Also, a method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is described. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division.

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The C/EBPalpha transcription factor is required for differentiation of adipocytes and neutrophil granulocytes, and controls cellular proliferation in vivo. To address the molecular mechanisms of C/EBPalpha action, we have identified C/EBPalpha mutants defective in repression of E2F-dependent transcription and found them to be impaired in their ability to suppress cellular proliferation, and to induce adipocyte differentiation in vitro. Using targeted mutagenesis of the mouse germline, we show that E2F repression-deficient C/EBPalpha alleles failed to support adipocyte and granulocyte differentiation in vivo. These results indicate that E2F repression by C/EBPalpha is critical for its ability to induce terminal differentiation, and thus provide genetic evidence that direct cell cycle control by a mammalian lineage-instructive transcription factor couples cellular growth arrest and differentiation.

To investigate the role of apoM in high density lipoprotein (HDL) metabolism and atherogenesis, we generated human apoM transgenic (apoM-Tg) and apoM-deficient (apoM ؊/؊ ) mice. Plasma apoM was predominantly associated with 10 -12-nm ␣-migrating HDL particles. Human apoM overexpression (11-fold) increased plasma cholesterol concentration by 13-22%, whereas apoM deficiency decreased it by 17-21%. The size and charge of apoA-I-containing HDL in plasma were not changed in apoM-Tg or apoM ؊/؊ mice. However, in plasma incubated at 37°C, lecithin:cholesterol acyltransferase-dependent conversion of ␣-to pre-␣-migrating HDL was delayed in apoM-Tg mice. Moreover, lecithin: cholesterol acyltransferase-independent generation of pre-␤-migrating apoA-I-containing particles in plasma was increased in apoM-Tg mice (4.2 ؎ 1.1%, p ‫؍‬ 0.06) and decreased in apoM ؊/؊ mice (0.5 ؎ 0.3%, p ‫؍‬ 0.03) versus controls (1.8 ؎ 0.05%). In the setting of low density lipoprotein receptor deficiency, apoM-Tg mice with ϳ2-fold increased plasma apoM concentrations developed smaller atherosclerotic lesions than controls. The effect of apoM on atherosclerosis may be facilitated by enzymatic modulation of plasma HDL particles, increased cholesterol efflux from foam cells, and an antioxidative effect of apoM-containing HDL.Epidemiological studies have demonstrated a strong inverse association between plasma HDL 3 concentrations and risk of premature coronary heart disease (1). However, the molecular heterogeneity of HDL has posed difficulties in understanding HDL metabolism and its effects in atherogenesis. In 1999, Xu and Dahlbäck (2) discovered apoM, which is mainly associated with HDL, with smaller amounts in LDL and VLDL. ApoM appears to have a novel role in murine HDL metabolism by affecting pre-␤-HDL formation (3). Pre-␤-HDL is considered antiatherogenic, because it mediates ATP-binding cassette transporter A1 (ABCA1)-dependent efflux of cholesterol from foam cells as part of the reverse cholesterol transport. Pre-␤-HDLs, the precursor of mature HDLs, are ϳ7-nm particles containing apoA-I and phospholipid and can be derived from two sources (4, 5). First, pre-␤-HDL can arise from newly synthesized hepatic apoA-I, acquiring free cholesterol and phospholipids through the interaction of apoA-I with ABCA1 (6). The small phospholipid-containing apoA-I particles can act via ABCG1 and remove cholesterol from foam cells (7-9). The free cholesterol in HDL can be esterified by lecithin:cholesterol acyltransferase (LCAT) (10, 11), changing the electrophoretic mobility of HDL from pre-␤ to ␣ (12-14). In mice, overexpression of LCAT converts ␣-HDL to pre-␣-HDL (15). Second, pre-␤-HDL can arise from ␣-HDL through the action of phospholipid transfer protein (PLTP), which induces fusion of ␣-HDL particles and concomitant dissociation of small pre-␤-HDL particles (16 -18). Also, hepatic lipase (HL) can generate pre-␤-HDL from ␣-HDL through its triglyceride lipase activity, either in concert with cholesteryl ester transfer protein (19) or alone (as in mice, w...

The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [ 125 I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink. Sparsomycin (Fig.