The MUC1 heterodimeric transmembrane glycoprotein is aberrantly overexpressed by diverse human carcinomas. Galectin-3 is a beta-galactoside binding protein that has also been associated with the development of human cancers. The present results demonstrate that MUC1 induces galectin-3 expression by a posttranscriptional mechanism. We show that the MUC1 C-terminal subunit is glycosylated on Asn-36 and that this modification is necessary for upregulation of galectin-3. N-glycosylated MUC1-C increases galectin-3 mRNA levels by suppressing expression of the microRNA miR-322 and thereby stabilizing galectin-3 transcripts. The results show that, in turn, galectin-3 binds to MUC1-C at the glycosylated Asn-36 site. The significance of the MUC1-C-galectin-3 interaction is supported by the demonstration that galectin-3 forms a bridge between MUC1 and the epidermal growth factor receptor (EGFR) and that galectin-3 is essential for EGF-mediated interactions between MUC1 and EGFR. These findings indicate that MUC1 and galectin-3 function as part of a miR-322-dependent regulatory loop.

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of ␣3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69 -88 amino acids) and T7 peptide (74 -98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via ␣ v ␤ 3 in an RGDindependent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

The soybean seed basic 7 s globulin (Bg) is capable of binding bovine insulin and insulin-like growth factors, and has protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor. A 4-kDa peptide named leginsulin, which can bind to Bg and compete with insulin for binding to Bg, was isolated from radicles of germinated soybean seeds. The leginsulin had a stimulatory effect on the phosphorylation activity of Bg, suggesting that it is involved in cellular signal transduction. The leginsulin was sequenced by automated Edman degradation and electrospray ionization mass spectrometry. It consisted of 37 amino acid residues with six half-cystines in three disulfide bridges. The mass spectrometric analysis revealed that a portion of the peptide is processed to delete the C-terminal glycine like a number of animal peptide hormones, but not C-terminally amidated. The cDNA encoding the leginsulin was cloned, sequenced and considered to code for a precursor polypeptide consisting of a putative signal peptide, the leginsulin, a linker peptide, a 6-kDa peptide and a C-terminal peptide. Although there is no sequence similarity between the leginsulin and insulin or insulin-like growth factors, the leginsulin is a possible candidate for plant peptide hormones.The involvement of insulin-like polypeptides in regulatory metabolic mechanisms in plants and microorganisms is still under question. However, there is some information available on insulin-binding proteins within these organisms. In yeast, a plasma membrane protein was found to be capable of binding mammalian insulin [ l , 21. Recently, we have isolated a protein from soybean which bound insulin and insulin-like growth factor and which was found to be a seed basic 7 s globulin (Bg) [3,4]. In some other legume species and in carrot, related proteins were also shown to bind bovine insulin and insulin-like growth factors [4], suggesting that insulin-binding proteins are widely distributed in many plant species.Although amino acid sequence similarity was found in a limited region between Bg and the human insulin-binding protein [51], no sequence similarity was found between Bg and the human insulin-or insulin-like growth factor receptor [6, 71. However, Bg showed structural similarities to the insulin receptor in glycosylation, the presence of a cysteinerich domain and disulfide-bonded a and p (27 kDa and 16 kDa in Bg) subunits. Both proteins are also synthesized as larger precursor polypeptides which are post-translationally cleaved at the N-terminal side of a serine residue to generate a and j? subunits. In Bg, we found a consensus sequence for a nucleotide-binding site, indispensable for protein phosphorylation [8], and a protein kinase activity which corresponds to about two thirds of the tyrosine kinase activity of the rat insulin receptor [9]. Furthermore, it was shown by immunocytochemistry that Bg is localized in the middle lamella of cell walls and the plasma membranes [lo]. These results suggest that Bg may...

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

Objective. To investigate the role of intercellular adhesion molecule 1 (ICAM-1) and ␤2 integrins in the production of superoxide (O 2 ؊ ) by C1q-stimulated human polymorphonuclear leukocytes (PMN).Methods. PMN were pretreated with F(ab) 2 fragments of monoclonal antibodies (mAb) that blocked or did not block ␤2 integrin-mediated adhesion. The cells were added to wells coated with C1q, and the production of O 2 ؊ was monitored kinetically as a color change due to reduction of cytochrome c. In some experiments, C1q was co-immobilized with purified ICAM-1.Results. Blocking mAb to the shared ␤2 integrin subunit, CD18, completely inhibited the O 2 ؊ response triggered by immobilized C1q, while blocking mAb to the ␣ subunits of the ␤2 integrins each partially blocked the O 2 ؊ response. PMN treated with C1q were found to activate the ␤2 integrins lymphocyte functionassociated antigen 1 and CR3 for binding to ICAM-1. Co-immobilization of ICAM-1 with C1q cooperatively triggered O 2؊ production by PMN. Conclusion. ␤2 integrin binding to an ICAM provided an essential costimulatory signal for O 2؊ production triggered by C1q in PMN. Our findings suggest a model for PMN activation in which 2 stimuli are required for O 2 ؊ production: a first signal that also activates PMN ␤2 integrins, followed by a second, ␤2 integrin-mediated signal, which occurs physiologically upon PMN binding to ICAM-1. The requirement for this dual signal for PMN generation of O 2 ؊ would serve as a regulatory mechanism to limit the production of O 2 ؊ to a tissue environment where C1q, or some other stimulus, is colocalized with stromal cells bearing upregulated ICAM-1. This mechanism may explain why all tissues can express ICAM-1 and may explain in part why inhibitors of tumor necrosis factor ␣, a major physiologic stimulus of ICAM-1 up-regulation, are potent antiinflammatory agents.

Mutual arrangement of histone H1 molecules in chromatin extended in low salt—EDTA buffer and additionally in the presence of urea was studied by means of reversible cross‐linking combined with chymotryptic digestion. In the chromatins tested, the chymotryptic halves of H1 were cross‐linked in all possible combinations; i.e., C—C, C—N and N—N. The results imply that the mutual arrangement of H1 histones is determined by the structure of extended nucleosomal chain, rather than chromatin superstructure.

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