Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

A novel cell surface antigen involved in thymocyte and thymic epithelial cell adhesion.

A murine mAb, 7D3, was produced by fusion of spleen cells obtained from mice immunized with a rat thymic epithelial cell line, Tu-D3 and NS/1 myeloma cells. 7D3 antibody reacted with approximately 95% thymocytes, 17% spleen cells, less than 9% of mesenteric lymph node cells and 32% of bone marrow cells of rat origin. 7D3 also reacted with two rat thymic epithelial cell lines but not with a rat fibroblastic cell line. Immunochemical analysis demonstrated that 7D3 antibody recognized a single polypeptide with molecular weight of 80,000 in FTE cells and 80,000 to 96,000 in thymocytes. 7D3 antibody strongly inhibited the thymocyte binding to thymic epithelial cells. In addition, 7D3 antibody inhibited TPA-induced thymocyte aggregation. 7D3 negative rat thymic lymphoma cells bound to 7D3 positive thymic epithelial cells and this binding was inhibited by 7D3 antibody, indicating that a part of thymocyte-thymic epithelial cell binding was mediated by the interaction of 7D3 Ag and undefined ligand to 7D3.     

The 55-kilodalton protein in an oriC complex fraction is glycogen synthase.

Three proteins with molecular masses of 35, 55, and 75 kDa were found in an oriC complex fraction after purification through CsCl density gradient centrifugation (W. G. Hendrickson, T. Kusano, H. Yamaki, R. Balakrishnan, M. King, J. Murchie, and M. Schaechter, Cell 30:915-923, 1982). Of these three proteins, the 55-kDa protein was determined to be glycogen synthase on the basis of the N-terminal amino acid sequence and the molecular weight. The oriC complex was formed in glgA mutant cells, which produce no detectable glycogen, as well as in wild-type cells. None of the 35-, 55-, and 75-kDa proteins were detected in the fraction from this mutant. The results indicate that these proteins were not constituents of the oriC complex.     

Thyrotropin-secreting pituitary adenoma: a case report.

We report a 44-year-old male with a thyrotropin (TSH)-secreting pituitary adenoma. Based serum free triiodothyronine (FT3, 12.1 pmol/l) and free thyroxine (FT4, 28 pmol/l) were increased with normal basal TSH (3.1 mU/l). There was impaired TSH response to thyrotropin releasing hormone (TRH) test. Serum TSH was suppressed to 59% of the basal level after oral administration of 1.4 mg 3,3'-5-triiodothyroacetic acid (triac), whereas no suppression was observed after 75 micrograms daily administration of triiodothyronine (T3). Serum concentrations of alpha-subunit of TSH (TSH-alpha) and TSH-alpha/TSH molar ratio were high, being 1.95 micrograms/l, and 4.4, respectively. Pituitary CT and MRI scan showed the presence of a macroadenoma in the anterior lobe of the pituitary gland. Histopathology of the excised pituitary confirmed the diagnosis of a TSH-producing adenoma. A positive correlation between TSH and FT3 (r = 0.66, P less than 0.01) or FT4 (r = 0.54, P less than 0.01) was observed in serial sera obtained before and after operation.     

Antiallergic mechanisms of beta-adrenergic stimulants in rats.

Antiallergic mechanisms of beta-adrenergic stimulants were investigated in rats. Isoproterenol administered intravenously inhibited IgE antibody-mediated homologous passive cutaneous anaphylaxis (PCA) and histamine-induced cutaneous reaction (HCR) elicited at the same time in the same rats significantly. The inhibition of PCA was more potent than that of HCR, suggesting that PCA is inhibited by at least 2 mechanisms. One is the inhibition of vascular permeability increase. In vivo histamine release in the rat peritoneal cavity caused by intravenous antigen was inhibited by the intravenous administration of isoproterenol or salbutamol dose-dependently. On the contrary, when the histamine release in the peritoneal cavity was caused by intraperitoneal antigen, isoproterenol or salbutamol administered simultaneously with antigen failed to inhibit the reaction. Furthermore, antigen-induced histamine release from sensitized peritoneal exudate cells in vitro was not inhibited by isoproterenol or salbutamol. These results indicate that the primary target of beta-adrenergic stimulants is the vascular endothelium, and that the direct inhibition of chemical mediator release from mast cells does not play an important role for the inhibition of PCA and in vivo histamine release in the peritoneal cavity in rats. Beta-adrenergic stimulants therefore may prevent intravenously administered antigen from activating sensitized mast cells through affecting endothelial cells.     

ATPase activity of SopA, a protein essential for active partitioning of F plasmid

Summary The SopA, B, C genes of the F plasmid play an essential role in plasmid partitioning during cell division in Escherichia coli. In this paper, the products of the sopA and sopB genes were isolated and their biochemical activities studied. [-³²P]ATP was cross-linked to the SopA protein by UV irradiation; this cross-linking was observed only in the presence of magnesium ion, and was competitively inhibited in the presence of non-radioactive ATP, ADP and dATP, but not other NTPs or dNTPs. In contrast, no ATP binding activity was detected for the SopB protein. The SopA protein showed a modest magnesium ion-dependent ATPase activity and this activity was stimulated in the presence of DNA. The ATPase activity in the presence of DNA was further stimulated by addition of the SopB protein. However, the SopB protein alone failed to stimulate the ATPase activity.     

Purification and characterization of histidinol dehydrogenase from cabbage

Histidinol dehydrogenase (EC 1.1.1.23) activity was determined in several plant species and in cultured plant cell lines. The enzyme was purified from cabbage (Brassica oleracea) to apparent homogeneity. To render complete purification, a new, specific histidinol-Sepharose 4B affinity chromatography was developed. The apparent molecular mass of the protein is 103 kDa. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein migrated as a single band with a molecular mass of 52 kDa, giving evidence for a dimeric quaternary structure. By isoelectric focusing, the enzyme was separated into six protein bands, five of which possessed the dehydrogenase activity when examined by an activity staining method. The Km values for L-histidinol and NAD+ were 15.5 and 42 microM, respectively. Enzyme activity was stimulated by addition of Mn2+, but was inhibited in the presence of Ba2+, Mg2+, Ni2+, Ca2+, Zn2+, or Cu2+. Histidinol dehydrogenase is the first histidine enzyme that has been purified to homogeneity and characterized from plants. This plant enzyme catalyzes the NAD-linked four-electron dehydrogenase reaction leading from histidinol to His. The results indicate a similar pathway of His in plants and show furthermore the last two reaction steps to be identical to those in microorganisms.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.     

Synthetic sialyl glycolipids (sialo-cholesterol and sialo-diglyceride) induce granulocytic differentiation of human myelogenous leukemia cell line HL-60

When HL-60 cells were cultivated with synthetic sialyl glycolipids, sialo-cholesterol and sialo-diglyceride, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was accompanied by inhibition of cell proliferation. The differentiation-inducing activity of sialo-cholesterol was greater than that of sialo-diglyceride on a molar basis, and the alpha-anomer of each compound was more potent than the beta-anomer, suggesting that the stereospecific structure of the compounds is important for the differentiation-inducing activity.