Macrophage colony stimulating factor: not just for macrophages anymore! A gateway into complex biologies.

Macrophage colony stimulating factor (M-CSF, also called colony stimulating factor-1) has traditionally been viewed as a growth/differentiation factor for monocytes, macrophages, and some female-specific tumors. As a result of alternative mRNA splicing and post-translational processing, several forms of M-CSF protein are produced: a secreted glycoprotein, a longer secreted form containing proteoglycan, and a short membrane-bound isoform. These different forms of M-CSF all initiate cell signaling in cells bearing the M-CSF receptor, called c-fms.

Gelsolin in complex with phosphatidylinositol 4,5-bisphosphate inhibits caspase-3 and -9 to retard apoptotic progression.

Apoptosis, or programmed cell death, occurs because of the activation of a protease cascade amplification circuit that includes the critical effector caspase-3. Previously, we identified the widely expressed actin modulatory protein gelsolin as a prominent substrate of caspase-3 and demonstrated that the N-terminal gelsolin cleavage product promotes apoptosis. Here we show that phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3, 4-bisphosphate in pure micelles or mixed vesicles prevent caspase-3 cleavage of gelsolin.

Structure-function studies on human macrophage colony-stimulating factor (M-CSF).

M-CSF (CSF-1) can be produced in a variety of structural forms that may affect function in vivo. Truncated, nonglycosylated forms of recombinant M-CSF (rM-CSF) from E. coli have been refolded in vitro in high yield and shown to be functionally equivalent in vitro to glycosylated rM-CSF secreted from mammalian cells.

Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion.

Galectin-3 is a beta-galactoside-specific lectin implicated in diverse processes involved in cellular interactions. Recently, the Mac-2-binding protein, a heavily N-glycosylated secreted protein with a subunit Mr of 97,000, was identified as its ligand. The present study characterizes the interaction between galectin-3 and Mac-2-binding protein in whole cells and measures their relative expression levels.

The crystal structure of PR3, a neutrophil serine proteinase antigen of Wegener's granulomatosis antibodies.

The crystal structure of PR3, a serine proteinase from the azurophilic granules of human polymorphonuclear neutrophils, has been solved by molecular replacement using the human leukocyte elastase structure. The PR3 structure has been refined to an R-factor (= sigma parallel Fo magnitude of-Fc parallel/sigma magnitude of Fo) of 0.201 for all data in the range of 10.

Recombinant proteins for medical use: the attractions and challenges.

Recombinant proteins that have survived the challenges of process development and clinical trials are becoming blockbuster medical products. Growth factors, enzymes and antibodies are being improved by mutational approaches, fused with other proteins, and even chemically modified in vitro. Drug development and testing approaches have advanced, and proteins produced in transgenic animals are new becoming available.

Structure-function studies on recombinant human macrophage colony-stimulating factor (M-CSF).

Human macrophage colony-stimulating factor (M-CSF) is a homodimeric cytokine that is a member of a structurally related family of hormones defined by an unusual up-up-down-down alpha-helical bundle. To identify regions on the surface of M-CSF that might interact with the M-CSF receptor, single and double amino acid substitutions were introduced into a truncated form of human M-CSF alpha by site-directed mutagenesis, and the homodimeric M-CSF analogs were purified and characterized. Certain substitutions in the region before and in helix A and in helix C decreased specific bioactivity and correlated with an approximately equivalent reduction in M-CSF receptor affinity.

Cloning and characterization of a human Mac-2-binding protein, a new member of the superfamily defined by the macrophage scavenger receptor cysteine-rich domain.

We have purified and sequenced a secreted glycoprotein from both the human breast carcinoma cell line, SK-BR-3, and human breast milk. The native protein binds specifically to a human macrophage-associated lectin known as Mac-2. This Mac-2 binding protein (Mac-2-BP) has an apparent native molecular mass of several million daltons and contains subunits of 85-97 kDa that are very susceptible to proteolysis at a dibasic cleavage site.

Three-dimensional structure of dimeric human recombinant macrophage colony-stimulating factor.

Macrophage colony-stimulating factor (M-CSF) triggers the development of cells of the monocyte-macrophage lineage and has a variety of stimulatory effects on mature cells of this class. The biologically active form of M-CSF is a disulfide-linked dimer that activates an intrinsic tyrosine kinase activity on the M-CSF receptor by inducing dimerization of the receptor molecules. The structure of a recombinant human M-CSF dimer, determined at 2.

Synthesis of the antibiotic cortalcerone from D-glucose using pyranose 2-oxidase and a novel fungal enzyme, aldos-2-ulose dehydratase.

Using two enzymes purified from the white-rot fungus, Polyporus obtusus, 5% solutions of D-glucose have been quantitatively converted in vitro into D-arabino-hexos-2-ulose (D-glucosone) and subsequently into a compound having antimicrobial activity. The antibiotic has been shown by nuclear magnetic resonance and mass spectroscopy to be chemically identical to a previously described fungal metabolite known as cortalcerone. Based on kinetic analysis of the synthetic process, a pathway for the biosynthesis of cortalcerone is proposed, involving both chemical rearrangement and enzymically catalyzed steps.

Purification, characterization, and western blot analysis of human GTPase-activating protein from native and recombinant sources.

Human ras GTPase-activating protein (GAP) is a cytoplasmic factor that stimulates the GTPase activity of normal N-ras p21 while having no stimulatory effect on the GTPase activity of oncogenic variants of N-ras p21. We have purified two forms of native ras GAP from human placental tissue. In addition to the Mr = 120,000 type I GAP reported previously (1), an equivalent amount of an Mr = 95,000 molecule with GAP activity was recovered and shown to have the N-terminal sequence expected for type II GAP.

Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing.

The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines.

Detection of endogenous macrophage colony-stimulating factor (M-CSF) in human blood.

We have detected endogenous human macrophage colony-stimulating factor (M-CSF) in blood of normal individuals, using a novel RIA that accurately measures M-CSF concentrations as low as 60 U/ml (1.2 ng/ml) in the presence of serum proteins. The RIA uses an antibody to highly purified recombinant human M-CSF and is calibrated to a mouse bone marrow colony-forming assay.

Molecular cloning of two types of GAP complementary DNA from human placenta.

The ras p21 GTPase-activating protein (GAP) was purified from human placental tissue. Internal amino acid sequence was obtained from this 120,000-dalton protein and, by means of this sequence, two types of complementary DNA clones were isolated and characterized. One type encoded GAP with a predicted molecular mass of 116,000 daltons and 96% identity with bovine GAP.

Recombinant human IL-2 overcomes genetic nonresponsiveness to malaria sporozoite peptides. Correlation of effect with biologic activity of IL-2.

Current malaria vaccine strategies focus on subunit vaccines that contain one or a limited number of malaria Ag. However, there is widespread nonresponsiveness to many of these Ag probably resulting from Ir gene control. Using a congenic mouse model, we demonstrated that human rIL-2 (as an adjuvant) can overcome Ir gene controlled low immune responsiveness to peptide malaria Ag vaccine candidates [R32tet32, R32LR, and Th2R-NP (NANP)5NA] as determined by the antibody response, providing it is emulsified with the Ag during immunization.

Reversed-phase chromatography of interleukin-2 muteins.

Human recombinant interleukin-2 (IL-2) and related species have been characterized by chemical modifications, tryptic digestion, and cyanogen bromide digestion. The oxidation states of the cysteines and methionines in several IL-2 muteins have been determined. Reversed-phase high-performance liquid chromatography allowed us to distinguish the modifications in these muteins and to correlate retention behavior with their structure.

Purified interleukin-2 induces proliferation of fresh human lymphocytes in the absence of exogenous stimuli.

Highly purified interleukin-2 (IL-2) induced proliferation of fresh human peripheral blood mononuclear leukocytes (PBML) in the absence of identifiable exogenous mitogenic or antigenic stimuli. Dose dependent proliferation was observed with three IL-2 preparations, including two preparations purified from natural sources and a preparation produced by recombinant DNA techniques. Both T and non-T cells proliferated.

Human recombinant interleukin-2 partly reconstitutes deficient in-vitro immune responses of lymphocytes from patients with AIDS.

The lymphokine interleukin-2 is required for the development of various cell-mediated immune functions that are known to be deficient in patients with acquired immunodeficiency syndrome (AIDS). The effects of pure human recombinant interleukin-2 (rIL-2), produced by Escherichia coli containing the cloned human gene, on in-vitro immune responses were studied in 16 patients with AIDS and 10 age-matched healthy heterosexual men. Exposure of lymphocytes from most AIDS patients to 1-100 U/ml rIL-2, increased mitogen and alloantigen induced proliferation and augmented natural killer (NK) cell function in a dose-dependent manner.

Biological activity of recombinant human interleukin-2 produced in Escherichia coli.

The gene for interleukin-2 was isolated from the Jurkat cell line and from normal peripheral blood lymphocytes and, when inserted in Escherichia coli, was expressed at high concentrations. This interleukin-2 was purified to apparent homogeneity and tested for biological activity in a variety of assays in vitro and in vivo. The recombinant lymphokine supports the growth of murine and human interleukin-2 dependent cell lines, enhances the generation of murine and human cytolytic cells in vitro, and generates lymphokine activated killer cells from murine and human lymphocytes.

The rolling circle . capsid complex as an intermediate in phi X DNA replication and viral assembly.

Late in the life cycle of the single-stranded DNA phage phi X, the synthesis of positive strand DNA is coupled to the maturation of progeny virions. DNA synthesis and packaging take place in a replication-assembly complex, which we have purified to homogeneity and characterized. The following conclusions can be drawn: 1.

Analysis of the phiX DNA replication cycle by electron microscopy.

We have monitored the development of intracellular phiX DNA forms during the course of a virus life cycle that duplicates as closely as possible the normal infection of individual cells by single virions. The viral DNA was isolated in a one-step purification procedure, and quantitative electron microscopy was performed on the samples, resulting in the following conclusions: (i) Early in the life cycle, when the cells accumulate duplex rings, two types of DNA replication intermediates are observed: a rolling circle with a single-stranded tail; and a novel form, a single-stranded circle that is partially duplex. Thus, duplex ring synthesis appears to occur in two asymmetric steps, with positive strand DNA first being processed from the tail of the rolling circle and circularized, before it acts as a template for negative strand synthesis.