Activated macrophages and antibodies against the plant lectin, GSI-B4, recognize the same tumor-associated structure (TAS)

Activated macrophages that were stabilized with either formalin or glutaraldehyde absorbed two polypeptides (Mr 100,000 and 60,000) from detergent extracts of all of the tumor cell lines tested, but not from detergent extracts of normal human peripheral blood lymphocytes. A major polypeptide (Mr 95,000) was retained from spent culture media of tumor cell lines. Polypeptides with molecular sizes of 100,000 and 60,000 daltons were also adsorbed by activated macrophages from detergent extracts of chicken embryo cell membranes, suggesting an oncofetal nature for these proteins. The 100,000 dalton polypeptide, but not the 60,000 dalton component, was found to be available to lactoperoxidase-catalyzed cell surface iodination. Polypeptides with identical molecular sizes could be adsorbed to immobilized galactopyranoside, indicating that they are vertebrate lectins. Activated macrophages and affinity adsorbents prepared by the covalent coupling of galactopyranoside to agarose also bind the plant lectin isolectin B4 prepared from the seeds of Griffonia simplicifolia. On the basis of these findings, we put forth the hypothesis that macromolecules of the same specificity, that is affinity to galactopyranosyl residues, must show homologies in their binding sites. We have predicted therefore that antisera prepared against this plant lectin should cross-react with galactopyranosyl-binding vertebrate lectins present on the surface of tumor cells. In this communication, we also report the generation of hybridomas that produce antibodies reactive with both the plant and vertebrate lectins. Inhibition experiments that make use of various mono- and disaccharides suggest that the specificities of these antibodies are for determinants intimately associated with the galactosyl binding site on the lectin molecule. Two of the antibodies were found to have moderate selectivity for tumor cells when tested in an immunohistochemical procedure that made use of fresh-frozen or paraffin-embedded sections of human biopsy material. These two antibodies on immunoblots of tumor cell membrane extracts reacted with a polypeptide with an apparent molecular size of 100,000 daltons.     

The stoichiometry of the Ca2+ pump in human erythrocyte vesicles: modulation by Ca2+, Mg2+ and calmodulin

Active Ca2+ uptake and the associated (Ca2+ + Mg2+)-ATPase activity were studied under the same conditions in an inside-out vesicle preparation of human red blood cells made essentially by the procedure of Quist and Roufogalis (Journal of Supramolecular Structure 6, 375-381, 1977). Some preparations were treated with 1 mM EDTA at 30 degrees to further deplete them of endogenous levels of calmodulin. As the Ca2+ taken up by the EDTA-treated inside-out vesicles, as well as the non-EDTA treated vesicles, was maintained after addition of 4.1 mM EGTA, the vesicles were shown to be impermeable to the passive leak of Ca2+ over the time course of the experiments. In the absence of added calmodulin, both active Ca2+ uptake and (Ca2+ + Mg2+)-ATPase were sensitive to free Ca2+ over a four log unit concentration range (0.7 microM to 300 microM Ca2+) at 6.4 mM MgCl2. Below 24 microM Ca2+ the stoichiometry of calcium transported per phosphate liberated was close to 2:1, both in EDTA and non-EDTA treated vesicles. Above 50 microM Ca2+ the stoichiometry approached 1:1. When MgCl2 was reduced from 6.4 mM to 1.0 mM, the stoichiometry remained close to 2:1 over the whole range of Ca2+ concentrations examined. In contrast to the results at 6.4 mM MgCl2, the Ca2+ pump was maximally activated at about 2 microM free Ca2+ and significantly inhibited above this concentration at 1 mM MgCl2. Calmodulin (0.5-2.0 microgram/ml) had little effect on the stoichiometry in any of the conditions examined. The possible significance of a variable stoichiometry of the Ca2+ pump in the red blood cell is discussed.     

Cytotoxic and antiprotozoal activity of flavonoids from Lonchocarpus spp.

A number of flavonoids isolated from Lonchocarpus spp. were evaluated for their antiprotozoal and cytotoxic activity. Flavone 6 and chalcone 7 were found to be the most active against Leishmania parasites and against cell cultures of Leukemia P388DI and adenocarcinoma prostate PC-3.     

Possible involvement of DNA breaks in epigenetic regulation of cell differentiation

The review summarizes the authors’ and literature data on accumulation of DNA breaks in differentiating cells. Large 50-kb free DNA fragments were observed by several research teams in non-apoptotic insect, mammal, and plant cells. More intense DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes, and neutrophils. In general, accumulation of DNA breaks in differentiating cells cannot be attributed to a decrease in the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulating the differentiation process. Scarce data on localization of the differentiation-associated DNA breaks indicate their preferable accumulation in specific DNA sequences including the nuclear matrix attachment sites. The same sites are degraded at early stages of apoptosis. Recent data on non-apoptotic function of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells, resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA breaks appears to be a prospective research direction.     

Selective diapedesis of Th1 cells induced by endothelial cell RANTES.

Differentiated CD4 T cells can be divided into Th1 and Th2 types based on the cytokines they produce. Differential expression of chemokine receptors on either the Th1-type or the Th2-type cell suggests that Th1-type and Th2-type cells differ not only in cytokine production but also in their migratory capacity. Stimulation of endothelial cells with IFN-gamma selectively enhanced transmigration of Th1-type cells, but not Th2-type cells, in a transendothelial migration assay. Enhanced transmigration of Th1-type cells was dependent on the chemokine RANTES produced by endothelial cells, as indicated by the findings that Ab neutralizing RANTES, or Ab to its receptor CCR5, inhibited transmigration. Neutralizing Ab to chemokines macrophage-inflammatory protein-1alpha or monocyte chemotactic protein-1 did not inhibit Th1 selective migration. Whereas anti-CD18 and anti-CD54 blocked basal levels of Th1-type cell adherence to endothelial cells and also inhibited transmigration, anti-RANTES blocked only transmigration, indicating that RANTES appeared to induce transmigration of adherent T cells. RANTES seemed to promote diapedesis of adherent Th1-type cells by augmenting pseudopod formation in conjunction with actin rearrangement by a pathway that was sensitive to the phosphoinositol 3-kinase inhibitor wortmannin and to the Rho GTP-binding protein inhibitor, epidermal cell differentiation inhibitor. Thus, enhancement of Th1-type selective migration appeared to be responsible for the diapedesis induced by interaction between CCR5 on Th1-type cells and RANTES produced by endothelial cells. Further evidence that CCR5 and RANTES play a modulatory role in Th1-type selective migration derives from the abrogation of this migration by anti-RANTES and anti-CCR5 Abs.