From a subtractive cDNA library» we isolated several cDNA clones which showed differential expression between highly and lowly metastatic human melanoma cell lines. One clone, desig nated nmb, showed preferential expression in the low-metastatic cell lines and was chosen for further characterization. Sequence analysis revealed that this clone represents a novel gene, encoding a putative transmembrane glycoprotein which showed the highest homology to the precursor of pMELI7, a melanocyte-specific protein. nmf> RNA expression was absent in most tumor-cell lines tested and not restricted to the melanocytic lineage. Transfection of a partial nmb cDNA into a highly metastatic melanoma cell line (BLM) resulted, in 2 of 3 transfectants, in slower subcutaneous tumor growth and, in I of 3 transfectants, in reduction of the potential for spontaneous metastasis in nude mice.

Activated leukocyte cell adhesion molecule (ALCAM/ CD166), a member of the immunoglobulin superfamily with five extracellular immunoglobulin-like domains, facilitates heterophilic (ALCAM-CD6) and homophilic (ALCAM-ALCAM) cell-cell interactions. While expressed in a wide variety of tissues and cells, ALCAM is restricted to subsets of cells usually involved in dynamic growth and/or migration processes. A structure-function analysis, using two monoclonal anti-ALCAM antibodies and a series of amino-terminally deleted ALCAM constructs, revealed that homophilic cell adhesion depended on ligand binding mediated by the membranedistal amino-terminal immunoglobulin domain and on avidity controlled by ALCAM clustering at the cell surface involving membrane-proximal immunoglobulin domains. Co-expression of a transmembrane ALCAM deletion mutant, which lacks the ligand binding domain, and endogenous wild-type ALCAM inhibited homophilic cell-cell interactions by interference with ALCAM avidity, while homophilic, soluble ligand binding remained unaltered. The extracellular structures of ALCAM thus provide two structurally and functionally distinguishable modules, one involved in ligand binding and the other in avidity. Functionality of both modules is required for stable homophilic ALCAM-ALCAM cell-cell adhesion.Adhesion molecules play an important role in development, leukocyte function, and homeostasis in multicellular organisms, which are mainly governed by inter-and intracellular communication via cell-cell interactions. Alterations in cellular adhesion and communication can contribute to uncontrolled cell growth (1) and life-threatening syndromes like leukocyte adhesion deficiency (2). Activation of adhesion molecules generally involves both modulation of affinity and avidity. The affinity of adhesion molecules often reflects a specific conformation of the extracellular ligand-binding domain. Avidity modulation involves changes in the cell surface distribution of adhesion molecules (e.g. lateral oligomerization), which leads to clusters of molecules and thereby specifically increases the number of available receptors at the site of cell-cell interaction.Activated leukocyte cell adhesion molecule (ALCAM/MEMD/ CD166) 1 is a type I transmembrane protein and a member of the Ig superfamily. It has over 90% homology with the chicken adhesion molecule BEN/SC1/DM-GRASP (3-5), and it has 30% identity and 50% similarity with the human melanoma cell adhesion molecule Mel-CAM/MUC18/CD146 (6). Furthermore, ALCAM has 93% sequence identity with the candidate liver high density lipoprotein receptor HB2 (7). ALCAM is involved in various physiological processes including hematopoiesis (8, 9), thymus development (10), the immune response (11), neurite extension (12), neural cell migration (13), and osteogenesis (14).ALCAM has a short cytoplasmic tail and its extracellular part comprises five Ig domains: two amino-terminal variable (V) type Ig domains followed by three constant (C) type Ig domains (V 1 V 2 C 1 C 2 C 3 ). ALCAM was first ident...

Mammals developed an immune system able to functionally polarize into so-called type 1 or type 2 immune pathways, to resolve infections with intracellular and extracellular pathogens, respectively. In the well-studied avian immune system of the chicken, however, no evidence for polarized immunity could be found, as yet. To investigate whether these two major arms of mammalian immunity, regulated by a T helper (Th)1/Th2 cytokine balance, evolved similarly in birds, chickens were exposed to a prevalent intracellular (viral) or extracellular (helminth) infection. By using semi-quantitative RT-PCR analysis we provide evidence that polarization of Th1/Th2 type immunity extends beyond mammalian species, and, therefore, has been evolutionary conserved for more than 300 million years, when the lineages of mammalian and avian vertebrates are assumed to have segregated.

During apoptosis, the U1-70K protein, a component of the spliceosomal U1 snRNP complex, is specifically cleaved by the enzyme caspase-3, converting it into a C-terminally truncated 40-kDa fragment. In this study, we show that the 40-kDa U1-70K fragment is still associated with the complete U1 snRNP complex, and that no obvious modifications occur with the U1 snRNP associated proteins U1A, U1C and Sm-B/B'. Furthermore, it is described for the first time that the U1 snRNA molecule, which is the backbone of the U1 snRNP complex, is modified during apoptosis by the specific removal of the first 5 ± 6 nucleotides including the 2,2,7-trimethylguanosine (TMG) cap. The observations that U1 snRNA cleavage is very specific (no such modifications were detected for the other U snRNAs tested) and that U1 snRNA cleavage is markedly inhibited in the presence of caspase inhibitors, indicate that an apoptotically activated ribonuclease is responsible for the specific modification of U1 snRNA during apoptosis. Cell Death and Differentiation (2000) 7, 70 ± 79.

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-γ synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.