Antigens associated with mammalian centromeres were localized at the light and electron microscopic levels using the peroxidase-labeled antibody method. The antibody used was of a type naturally occurring in the sera of patients with scleroderma . At the light microscopic level, it reacts specifically with the centromere regions of chromosomes in a variety of mammalian species and strains in discrete foci in interphase nuclei . We find that the number of foci approximates the number of chromosomes present in the various cell types . At the ultrastructural level, the antigenic foci are confirmed to lie in the kinetochore regions of each chromosome . In interphase nuclei, the antigenic foci were usually associated either with the inner surfaces of the nuclear envelope or with the nucleoli. These observations indicate that the centromere regions of the chromosomes in interphase are not randomly distributed within the nucleus but are usually fixed either to the inner surface of the nuclear envelope or to nucleoli .
The mammalian genome contains 20-30 genes encoding a family of actins. To date, however, only six proteins (four muscle and two nonmuscle isoforms) encoded by this multigene complex have been identified. We have isolated two actins from the brush border of rat intestinal epithelial cells that have isoelectric points and N-terminal peptides characteristic of the cytoplasmic beta- and gamma-actins. However, using a panel of actin-specific monoclonal antibodies, we show that these actins contain a set of epitopes that distinguishes them from any of the known cytoplasmic or muscle isoforms. These unique actins share features of both the nonmuscle and muscle isoforms, suggesting that they represent an intermediate in the evolution of the specialized muscle actins.
Experiments were conducted to determine whether a partially purified listeria cell wall fraction could stimulate macrophages to high levels of activation. To detect activation of macrophages, a macrophage-mediated cytotoxicity system were established. The data demonstrate that listeria cell wall components are capable of activating thioglycollate-induced adherent peritoneal exudate cells to be cytotoxic for 51Cr-labelled target tumour cells, and that the listeria fraction is as effective as bacterial lipopolysaccharide in inducing cytotoxicity. The listeria fraction can also induce peritoneal exudate cells from congenitally thymusless nude mice to become cytotoxic, suggesting that mature T cells are not required. Furthermore, thioglycollate-induced adherent peritoneal exudate cells from mice hyperimmunized to live Listeria organisms are already stimulated to be cytotoxic for tumour cells, and do not need to be activated in vitro. Additional data are presented which characterize the system. These data demonstrate that a critical concentration of adherent peritoneal cells is required for in vitro activation. Moreover, only peritoneal cells induced with aged batches of thioglycollate, and not uniduced peritoneal cells or those induced with fresh thioglycollate or with protease peptone can be activated in vitro to kill tumour cells. Evidence is presented which suggests that the cytotoxic cell is a macrophage.
A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.
T-cells are an integral part of normal follicular centers and many follicular center-cell (FCC) lymphomas. Because the functional role of these cells remains imprecisely determined and because ultrastructural localization of the T-cell antigen receptor-associated CD3 antigen has not been previously reported, an immunoultrastructural study of four tonsils and four FCC lymphomas was performed using an anti-CD3 antibody (UCHT-1). Normal interfollicular CD3-positive T-cells always demonstrated surface membrane positivity, as did 94% of normal and 88% of neoplastic FCC-associated CD3-positive cells. Conversely, whereas only 5% of normal interfollicular CD3-positive cells showed perinuclear positivity, 58% of normal and 38% of neoplastic FCC-associated CD3-positive cells did (p less than 0.001). Definite endoplasmic reticulum (ER) staining for CD3 was identified in 4% and 8% of cells associated with normal or neoplastic FCC, respectively, but in none of the T-zone lymphocytes. Because the perinuclear space is reported to be a site of protein synthesis in cells with little ER, and because of the occasional ER staining observed in cells with perinuclear staining, perinuclear CD3 positivity probably represents CD3 synthesis in mature tonsillar T-cells. The frequency of perinuclear positivity in the FCC-associated T-cells, together with the loss of surface positivity in some cells at this site, suggests that this could represent in vivo T-cell "activation" in follicular centers, with modulation and resynthesis of the CD3 antigen. Furthermore, the results demonstrate a similar phenomenon in the T-cells associated with neoplastic follicular center cells.
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