Macrophages play a key role in both normal and pathological processes involving immune and inf lammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor ␤ (TGF-␤) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1␤, tumor necrosis factor ␣ (TNF-␣), interleukin 2, and macrophage colony-stimulating factor but not interferon ␥, or lipopolysaccharide (LPS). Its expression is also increased by TGF-␤. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of M r 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-␣ production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinf lammatory cytokines and TGF-␤ may serve to limit the later phases of macrophage activation.

Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles. However, until recently they were believed not to occur at the nuclear membrane. In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937. NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles. NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.Whereas ion channels have been found on the membranes of various intracellular organelles, it has only been recently that patch clamping studies have suggested their existence at the nuclear membrane. The nuclear pore complexes have been considered the site of communication and exchange between the nucleus and cytoplasm (1, 2). Studies of traffic across the nuclear envelope have in general conformed to the paradigm that ions and small metabolites with diameters of less than 3-4 nm passively diffuse across the nuclear envelope (3). Thus, the concept of nuclear membrane ion channels seems at variance with the generally accepted views of the nuclear envelope, with their function at this location seeming redundant. However, this does not appear to be the case.The first demonstration of ionic conductances in the nuclear membrane were in mouse zygote pronuclei (4). Further evidence then followed with the demonstration of ion-selective channels in avian erythrocytes (5), in mouse oocyte germinal vesicles, in nuclei from two-cell embryos and liver (6), in the nuclei of cardiac myocytes (7), and in rat hepatocyte nuclei (8). Mak and Foskett (9) described the presence of inositol 1,4,5-trisphosphate-dependent receptor channels in isolated nuclei which were activated by inositol 1,4,5-trisphosphate, inhibited by heparin and selective to calcium ions. Similarly, Pasyk and Foskett (10), have shown chloride channel activity in isolated nuclei from CHO 1 cells. More recently, following fractionation and reconstitution of inner and outer nuclear membrane fractions into lipid bilayers, Rousseau et al. (11) have shown the presence of two types of chloride channels. The use of calcium ion imaging techniques has also demonstrated variations in calcium ion concentrations between the nucleus and cytoplasm, again suggesting a selective uptake or retention of these ions by the nucleus (12, 13).With growing electrophysiological data for the existence of nuclear ion channels, it is clear that the cloning and isolation of these proteins will greatly assist in determining their structure and function. This paper describes the molecular cloning and characterization of what we believe to be the first chloride ion channel protein of the nuclear membrane and only the second cloned ion chan...

The Dictyostelium discoideum cell surface antigen PsA is a glycoprotein which first appears in the multicellular stage soon after tip formation and is selectively expressed on prespore cells. The D19 gene encodes an mRNA sequence which is highly enriched in prespore over prestalk cells in the slug stage. We have determined 81 amino acid residues of N-terminal sequence from immunoaffinity-purified PsA protein and shown this sequence to be identical to the predicted sequence of the D19 gene. There are several short repeat elements close to the C terminus, and unequal crossing-over within these is proposed to account for the size polymorphism observed in PsA protein isolated from different D. discoideum strains. The repeats are proline rich and show similarity to the C-terminal region of the D. discoideum cell adhesion molecule, contact sites A. The extreme C terminus, which is also homologous to contact sites A, is characteristic of proteins attached to the plasma membrane via a glycosyl-phosphatidylinositol link. We have marked the PsA gene by insertion of an oligonucleotide encoding an epitope of the human c-myc protein. A construct containing this gene and 990 base pairs of 5'-flanking region directed correct temporal and spatial mRNA accumulation. We found the marked PsA protein, detected with the human c-myc antibody, to be correctly localized on the surface of cells.